METHODS OF TREATING CANCER

METHODES DE TRAITEMENT DU CANCER

English Abstract

Provided herein are, inter alia, methods of treating cancer by administering to a subject a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist or a combination of an adenosine-A2A(A2A)receptor antagonist and a programmed cell death protein 1(PD-1) signaling pathway inhibitor. Further provided are pharmaceutical compositions including an A2Areceptor antagonist, a PD-1 signaling pathway inhibitor and a pharmaceutically acceptable excipient. Further provided are methods of detecting cellular effects,for example expression of pCREB, before, after or during adenosine receptor antagonist treatment.

French Abstract

L'invention concerne, <i />entre autres, des méthodes de traitement du cancer par administration au patient d'une quantité thérapeutiquement efficace d'un antagoniste du récepteur A2A de l'adénosine (A2A) ou d'une combinaison d'un antagoniste du récepteur A2A de l'adénosine (A2A) et d'un inhibiteur de la voie de signalisation de la protéine de mort cellulaire programmée 1 (PD-1) . L'invention concerne en outre des compositions pharmaceutiques comprenant un antagoniste du récepteur A2A, un inhibiteur de la voie de signalisation de PD-1 et un excipient pharmaceutiquement acceptable. L'invention concerne en outre des méthodes permettant de détecter des effets cellulaires, par exemple l'expression de pCREB, avant, après ou pendant un traitement utilisant un antagoniste du récepteur de l'adénosine.

Claims

84342314
CLAIMS:
1. Use of an adenosine-A2A receptor antagonist of formula:
\ --N
N
0
NH2 or a pharmaceutically acceptable
salt thereof, to treat colon cancer, lung cancer, triple negative breast
cancer, melanoma, head and
neck cancer, prostate cancer, bladder cancer, renal cell carcinoma, colorectal
cancer, or
lymphoma; wherein the adenosine-A2A receptor antagonist is for administration
in an amount of
about 100 mg twice a day.
2. Use of an adenosine-A2A receptor antagonist of formula:
\ N
0
0
0
N112 or a pharmaceutically
acceptable salt thereof, to increase an anti-tumor immune response in a
subject having colon
cancer, lung cancer, triple negative breast cancer, melanoma, head and neck
cancer, prostate
cancer, bladder cancer, renal cell carcinoma, colorectal cancer, or lymphoma;
wherein the
adenosine-A2A receptor antagonist is for administration in an amount of about
100 mg twice a day.
3. Use of an adenosine-A2A receptor antagonist of formula:
204
Date recue/Date received 2023-04-05

84342314
\ N
N --
1
0
0
0 N
NH2 or a pharmaceutically
acceptable salt thereof, to decrease tumor volume in a subject having colon
cancer, lung cancer,
triple negative breast cancer, melanoma, head and neck cancer, prostate
cancer, bladder cancer,
renal cell carcinoma, colorectal cancer, or lymphoma; wherein the adenosine-
A2A receptor
antagonist is for administration in an amount of about 100 mg twice a day.
4. Use of an adenosine-A2A receptor antagonist of formula:
--N
N
0
0
0 N
N}12 or a pharmaceutically
acceptable salt thereof, to enhance anti-tumor immune memory in a subject
having colon cancer,
lung cancer, triple negative breast cancer, melanoma, head and neck cancer,
prostate cancer,
bladder cancer, renal cell carcinoma, colorectal cancer, or lymphoma; wherein
the adenosine-A2A
receptor antagonist is for administration in an amount of about 100 mg twice a
day.
5. The use of any one of claims 1 to 4, further comprising use of
atezolizumab to
treat the cancer.
6. The use of claim 5, wherein the atezolizumab is for administration in an
amount
of about 840 mg.
7. The use of claim 5, wherein the atezolizumab is for administration in an
amount
of about 1200 mg.
8. The use of any one of claims 5 to 7, wherein the atezolizumab is for
administration once every two weeks.
205
Date recue/Date received 2023-04-05

84342314
9. The use of any one of claims 5 to 7, wherein the atezolizumab is for
administration once every three weeks.
10. The use of any one of claims 1 to 9, wherein the cancer is renal cell
carcinoma.
11. The use of any one of claims 1 to 9, wherein the cancer is triple
negative breast
cancer.
12. The use of any one of claims 1 to 9, wherein the cancer is melanoma.
13. The use of any one of claims 1 to 9, wherein the cancer is head and
neck cancer.
14. The use of any one of claims 1 to 9, wherein the cancer is prostate
cancer.
15. The use of any one of claims 1 to 9, wherein the cancer is bladder
cancer.
16. The use of any one of claims 1 to 9, wherein the cancer is colorectal
cancer.
17. The use of any one of claims 1 to 9, wherein the cancer is lymphoma.
18. The use of any one of claims 1 to 9, wherein the cancer is colon
cancer.
19. The use of any one of claims 1 to 9, wherein the cancer is lung cancer.
20. The use of claim 19, wherein the lung cancer is non-small cell lung
cancer.
21. The use of any one of claims 1 to 20, wherein the subject is an anti-PD-
1
refractory subject.
22. The use of any one of claims 1 to 21, wherein the cancer is a primary
cancer.
23. The use of any one of claims 1 to 22, wherein the adenosine-A2A
receptor
antagonist is 7-(5-methylfuran-2-y1)-34[6-[[(3S)-oxolan-3-ylloxymethyllpyridin-
2-
yljmethylltriazolo[4,5-d]pyrimidin-5-amine or a pharmaceutically acceptable
salt thereof.
24. Use of atezolizumab and an adenosine-A2A receptor antagonist of
foimula:
206
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84342314
\ ,N
N
1
0
0
0
NI-12
or a pharmaceutically acceptable salt thereof to treat cancer; wherein the
cancer is colon cancer,
lung cancer, triple negative breast cancer, melanoma, head and neck cancer,
prostate cancer,
bladder cancer, renal cell carcinoma, colorectal cancer, or lymphoma.
25. The use of claim 24, wherein the adenosine-A2A receptor antagonist is
for
administration at 100 mg.
26. The use of claim 24 or 25, wherein the adenosine-A2A receptor
antagonist is for
administration twice a day.
27. The use of any one of claims 24 to 26, wherein the atezolizumab is for
administration in an amount of about 840 mg.
28. The use of any one of claims 24 to 26, wherein the atezolizumab is for
administration in an amount of about 1200 mg.
29. The use of any one of claims 24 to 28, wherein the atezolizumab is for
administration once every two weeks.
30. The use of any one of claims 24 to 28, wherein the atezolizumab is for
administration once every three weeks.
31. The use of any one of claims 24 to 30, wherein the cancer is renal cell
carcinoma.
32. The use of any one of claims 24 to 30, wherein the cancer is triple
negative breast
cancer.
33. The use of any one of claims 24 to 30, wherein the cancer is melanoma.
34. The use of any one of claims 24 to 30, wherein the cancer is head and
neck
cancer.
207
Date recue/Date received 2023-04-05

84342314
35. The use of any one of claims 24 to 30, wherein the cancer is prostate
cancer.
36. The use of any one of claims 24 to 30, wherein the cancer is bladder
cancer.
37. The use of any one of claims 24 to 30, wherein the cancer is colorectal
cancer.
38. The use of any one of claims 24 to 30, wherein the cancer is lymphoma.
39. The use of any one of claims 24 to 30, wherein the cancer is colon
cancer.
40. The use of any one of claims 24 to 30, wherein the cancer is lung
cancer.
41. The use of claim 40, wherein the lung cancer is non-small cell lung
cancer.
42. The use of any one of claims 24 to 41, wherein the subject is an anti-
PD-1
refractory subject.
43. The use of any one of claims 24 to 42, wherein the cancer is a primary
cancer.
44. The use of any one of claims 24 to 43, wherein the adenosine-A2A
receptor
antagonist is 7-(5-methylfuran-2-y1)-34[6-[[(3S)-oxolan-3-yl]oxymethyl]pyridin-
2-
yl]methylltriazolo[4,5-dlpyrimidin-5-amine or a pharmaceutically acceptable
salt thereof.
208
Date recue/Date received 2023-04-05

Description

84342314
METHODS OF TREATING CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Application No. 62/387,383,
filed December
24, 2015, U.S. Application No. 62/324,211, filed April 18, 2016, U.S.
Application No.
62/350,602, filed June 15, 2016, U.S. Application No. 62/421,109, filed
November 11, 2016, and
U.S. Application No. 62/421,171, filed November 11, 2016.
BACKGROUND OF THE INVENTION
[0002] The goal of immunotherapy is to drive cytotoxic T-cell responses to
eradicate cancer.
To prevent reaction to self-antigens, or overreaction, multiple inhibitory
checkpoint signals exist
including PD1/2, CTLA4 and adenosine. Extracellular adenosine, a purine
nucleoside, is
produced during acute, inflammatory processes by conversion from adenosine
triphosphate
(ATP) through ectonucleotidases CD73 and CD39 expressed on the cell surface of
multiple
tissue types. Adenosine is normally upregulated to protect a host from over-
injury in response to
such stimuli as infection or ischemia by binding its extracellular, G-protein
coupled receptors on
target cells (including AIR, A2AR, A2BR, and A3R) and begin healing {Hasko
2008}.
However, multiple tumor types can actively sustain extracellular adenosine
levels well beyond
acute phase reactions to dampen a host's immune response through multiple
mechanisms
{Antionioli 2013}. Increases in adenosine in the microenvironment by malignant
cells recruits
regulatory T-cells (Treg),which express substantial CD39, to the area and
further drive up
adenosine levels {Sica 2010}.
[0003] Cancer cells also appear to directly utilize adenosine. As a result,
adenosine causes
inefficient presentation of tumor antigens to the adaptive system and enhances
tumor growth.
Thus, there is a need in the art for effective cancer treatments. The methods
and compositions
provided herein address these and other deficiencies in the art.
BRIEF SUMMARY OF THE INVENTION
[0004] In one aspect, a method of treating cancer in a subject in need thereof
is provided. The
method includes administering to the subject a therapeutically effective
amount of an adenosine-
1
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WO 2017/112917 PCT/US2016/068459
A2A (A2A) receptor antagonist and a programmed cell death protein 1 (PD-1)
signaling pathway
inhibitor.
[0005] In another aspect, a method of treating cancer in a subject in need
thereof is provided.
The method includes administering to the subject a therapeutically effective
amount of an
adenosine-A2A (A2A) receptor antagonist of formula:
RI
N,
N 2
(1).
[0006] In formula (I), RI is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -S0,11R9,
-SO,INR9R1 , -NHNH2, -0NR912.1 , -NHC=(0)NHNH2, -NHC-(0)NR9- _
N(0)mi,-NR912.1 ,
-C(0)R9, -C(0)-0R9, -C(0)NR9Rio, _OR9, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0n2R11,
-S0,2NRI1R12, NHNH2, 0NRI1R12, _(0)NHENH2, _(0)NRI1R12, _N(0)m2,
_NR1iRt2, -NH-O-R", -C(0)R",
-C(0)-OR", -C(0)NRItRi2, _0-K.11, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -S0n3R13,
-S0,3NR ONR13R14,
NHC-(0)NHNH2, -NHC=(0)NR13R14, _N(0)m3,

_NR13R14, _NH-O-R13, _coy.K 13,
- C(0)-0R13, -
C(0)NRI3R14, _0-13, =
substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, Rio, Rn, R12, Ri3 and R'4
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
SO4H,
-SO2NH2, -NO2, -NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or
unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
2

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heteroaryl. Xa, Xb and Xc are independently -F, -Cl, -Br, or -I. The symbols
ni, n2 and n3 are
independently an integer from 0 to 4. The symbols ml, m2 and m3 are
independently an integer
from 1 to 2. And the symbols vi, v2 and v3 are independently an integer from 1
to 2.
[0007] In one aspect, a method of activating a T cell is provided. The method
includes
contacting the T cell with an A2A receptor antagonist, wherein the A2A
receptor antagonist is a
compound of formula:
Na
(1).
[0008] In formula (I), RI is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -SO11R9,
-SO,INR9Rth, _NHNH2, _0NR9Rio, _NHc="NHNH2, _NHc="NR9Rio, _N(0).1,_NR9Rio,
-C(0)R9, -C(0)-0R9, -C(0)NR9R1 , -0R9, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0,12R",
-S0v2NR11R12, _NHNH2, _oNRitR127 _NHc_oz,NHNH2, _Ntic _(0)NRI1R12, _N(0)1

i27
_NR11R12, -NH-O-R", _c(or
K -C(0)-OR", -C(0)NRIIR12, _0-11, substituted or
unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CX'3, -CN, -
S02C1, -S0,13R13,
-S0,3NRI3R14, _NHNH2, _coNRI3R14, _NHc="NHNH2, _NHc="NRi3R14, _N(0).3,
_NH_O_Ri3, -C(0)R'3,
C(0)-0R13, -C(0)NRi3R14,
substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, RI , RH, Ru, R13 and R14
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
SO4H,
3

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-SO2NH2, -NO2, -NH2, -NHNH2,
-NHC=(0)NHNI-12, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. xa,Xb and X' are independently -F, -Cl, -Br, or -I. The symbols
n1, nz and n3 are
independently an integer from 0 to 4. The symbols ml, mz and m3 are
independently an integer
from 1 to 2. The symbols v1, vz and v3 are independently an integer from 1 to
2.
[0009] In one aspect, a method of inhibiting A2A receptor activity of a cell
is provided. The
method includes contacting the cell with an A2A receptor antagonist, wherein
the A2A receptor
antagonist is a compound of formula:
N,
2
R3 (0.
[0010] In formula (I), R1 is independently hydrogen, halogen, -C303, -CN, -
S02C1, -S011,R9,
-SOvINR9Rio, _NHNH2, _0NR9Rio, _NHc=.(0)NHNH2, _NHc=(0)NR9Rio,
_N(0).1,_NR9Rio,
-C(0)R9, -C(0)-0R9, -C(0)NR9R1o, -OR9, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
SOraltll,
-S0v2NRIIR12, miNH2, 0NRI1R12, Nac _(0)NHNH2, NHIc _(0)NRI1R12, _N(0).2,
-NR
C(0)-0R11, -C(0)NRitRi2, -OR", substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CX`3, -CN, -
S02C1, -S0,13R13,
-S0v3NRI3R14, _NHNH2, _0NRI3R14, _NHc_(0)NHNH2, _Nuc_(0)NR13RI4, _N(0).3,
-NR13R14, -NH-O-R13, -C(0)R13, -C(0)-0R13, -C(0)NR13R14,
substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
4

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unsubstituted heteroaryl. R9, Rio, Ri2, Ri3 and R'4
are independently hydrogen, halogen,
=0, -S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
S0411,
-SO2NH2, -NO2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. Xa, Xb and Xc are independently -F, -Cl, -Br, or -I. The symbols
n1, n2 and n3 are
independently an integer from 0 to 4. The symbols m I, m2 and m3 are
independently an integer
from 1 to 2. The symbols vi, v2 and v3 are independently an integer from 1 to
2.
[0011] In one aspect, a method of increasing an anti-tumor immune response in
a subject in
need thereof is provided. The method includes administering to the subject a
therapeutically
effective amount of an adenosine-A2A (A2A) receptor antagonist and a
programmed cell death
protein 1 (PD-1) signaling pathway inhibitor.
[0012] In another aspect, a method of increasing an anti-tumor immune response
in a subject
in need thereof is provided. The method includes administering to the subject
a therapeutically
effective amount of an adenosine-A2A (A2A) receptor antagonist, wherein the
A2A receptor
antagonist is a compound of formula:
RI
Na
2
(I).
[0013] In formula (I), RI is independently hydrogen, halogen, -CX53, -CN, -
S02C1, -SOn1R9,
-S0,1NR9R1 , -NHNH2, -0NR9R1 , -NI-10---(0)NHNE12, -NHC-(0)NR9R1 , -N(0).11,-
NR9Rio,
-C(0)R9, -C(0)-0R9, -C(0)NR9R ,to -0R9, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0n2R11,
-S0,2NRI iRi2, r.sar,s4-H2, 0NR t iR12, _(0)NHN-H2, _(0)NR
R12, _N(0)m2,

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_C(0)-OR", -C(0)NR' 'R'2, , =
sunstauted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CX'3, -CN, -
S02C1, -S0113R13,
-S0,3NRI3R14, _NHNH2, _0NRI3R14, _NHc=(0)NHNH2, _NHc=(0)NRI3R14, _N(0).3,
_NRi3R14, _NH_O_Ri3, _c(0)R13, _
C(0)-0R13, -C(0)NR13R14, _0R13, ,
sunstituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, Rio, Ri2, Ri3 and R'4
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CON112, -OH, -SH, -S02C1, -S03H, -
SO4H,
-SO2NI-12, -NO2, -NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or
unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. xa, Xb and Xc are independently -F, -Cl, -Br, or -I. The symbols
n1, nz and n3 are
independently an integer from 0 to 4. The symbols ml, m2 and m3 are
independently an integer
from 1 to 2. The symbols v1, vz and v3 are independently an integer from 1 to
2.
[0014] In one aspect, a method of increasing the amount of CD8-positive cells
relative to the
amount of regulatory T cells in a subject in need thereof is provided. The
method includes
administering to the subject a therapeutically effective amount of an
adenosine-A2A (A2A)
receptor antagonist and a programmed cell death protein 1 (PD-1) signaling
pathway inhibitor.
[0015] In one aspect, a method of increasing the amount of CD8-positive cells
relative to the
amount of regulatory T cells in a subject in need thereof is provided. The
method includes
administering to the subject a therapeutically effective amount of an
adenosine-A2A (A2A)
receptor antagonist, wherein the A2A receptor antagonist is a compound of
formula:
RI
\N R 2
R3 (1).
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[0016] In formula (I), le is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -S0,11R9,
-SO,INR9Rio, _NHNH2, _0NR9Rio, _NHc=(0)NHNH2, _NHc=(0)NR9Rio, _
N(0)õ,i,-
NR9Rio,
-C(0)R9, -C(0)-0R9, -C(0)NR9R1 , -0R9, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0n2R11,
-S0,2NRI1R12, __NHNH2, _0NRI1R12, NHc =(0)NHNT-H2, =(0)NRIIR12, _N(c)m2,
-C(0)-OR", -C(0)NR11R12,
-
K substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. le is independently hydrogen, halogen, -CX'3, -CN, -
S02C1, -SOõ3R13,
S0,3NR13R14, _NHNH2, _0NRI3R14, _NHc=o)NHNH2, _NHc=o)NRI3Ri4, _N(0).3,
-
_NR13R14, _c(o)R13, _
C(0)-0R13, -C(0)NRI3e, , ,
- suostitutea or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, Rio, RI% R12, R13 and K-14
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
S041-1,
-SO2NH2, -NO2, -NH2, -NINFI2, -ONH2, -NHC-(0)NHNF!2, substituted or
unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. Xa, Xb and X' are independently -F, -Cl, -Br, or -I. The symbols
n1, n2 and n3 are
independently an integer from 0 to 4. The symbols ml, m2 and m3 are
independently an integer
from 1 to 2. The symbols vi, v2 and v3 are independently an integer from 1 to
2.
[0017] In one aspect, a method of decreasing tumor volume in a subject in need
thereof is
provided. The method includes administering to the subject a therapeutically
effective amount of
an adenosine-A2A (A2A) receptor antagonist and a programmed cell death protein
1 (PD-1)
signaling pathway inhibitor.
[0018] In one aspect, a method of decreasing tumor volume in a subject in need
thereof is
provided. The method includes administering to the subject a therapeutically
effective amount of
7

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an adenosine-A2A (A2A) receptor antagonist, wherein the A2A receptor
antagonist is a
compound of formula:
RI
\N
[0019] In formula (I), le is independently hydrogen, halogen, -CXa3, -CN, -
S02C1, -S0111R9,
-SOvi.NR9Rio, _NHNH2, _0NR9Rio, _NHc=(0)NHNH2, _NHc=(0)NR9Rio,
_N(0).1,_NR9Rio,
-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9R1o, -0R9, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0,12R11,
-SC:0,2NR' IR12, NHNH2, 0NR tRu, NHic (0)NHNH2, NHic (0)NR"Ru, _N(0).2,
-C(0)R", -c(0)-OR", -C(0)NR" R'2, -OR", substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CX'3, -CN, -
S02C1, -S0n31e3,
-S0,3NRI3R14, _NHNH2, _0NRI3R14, _NHc="NHNH2, _NHc=(0)NRi3R14,
_Nee, _NH_O-R13, -C(0)R'3,
C(0)-OR13, -C(0)NR13R14, -0R'3, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, Ru:), Rn, Ru, R13 and K-14
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
SO4H,
-SO2NH2, -NO2, -NHNH2, -0N1-12, -NHC=(0)NHNH2, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. Xa, Xb and X' are independently -F, -Cl, -Br, or -I. The symbols
ni, nz and n3 are
independently an integer from 0 to 4. The symbols mi, mz and m3 are
independently an integer
from 1 to 2. The symbols vl, vz and v3 are independently an integer from 1 to
2.
8

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[0020] In one aspect, a method of enhancing anti-tumor immune memory in a
subject in need
thereof is provided. The method includes administering to the subject a
therapeutically effective
amount of an adenosine-A2A (A2A) receptor antagonist and a programmed cell
death protein 1
(PD-1) signaling pathway inhibitor.
[0021] In one aspect, a method of enhancing anti-tumor immune memory in a
subject in need
thereof is provided. The method includes administering to the subject a
therapeutically effective
amount of an adenosine-A2A (A2A) receptor antagonist, wherein the A2A receptor
antagonist is
a compound of formula:
RI
R3 (1).
[0022] In formula (I), RI is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -SOniR9,
_NHNH2, _0NR9Rio, _NHc=(0)NHNH2, _NHc=(0)NR9Rio, _N(0)mi,_NR9Rio,
-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9R1 , -0R9, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -S0R11,
-S0,2NRIIR12, _NH1NH2, _0NRiiR12, _
NHC-(0)NHNH2, -NHC-(0)NR11R12, 4s1(0)1n2,
_NRIIR12, -C(0)R", _C(0)-0R11, -C(0)NR"R12, _0-11, =
sunstautea or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CX'3, -CN, -
S02C1, -S0n3R13,
-S0,3NR13R14, _NHNH2, _0NR13R14, _NHc=(0)NHNH2, _NHc=(0)NR13R14, _N(0)m3,
-NRI3R14, _NH_O-R13, _c(0)R13, _
C(0)-0R13, -C(0)NRI3R14, _0-13, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
9

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unsubstituted heteroaryl. R9, Ru:), Rn, R12, R13 and R'4
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
S0411,
-SO2NH2, -NO2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. Xa, Xb and Xc are independently -F, -Cl, -Br, or -I. The symbols
n1, n2 and n3 are
independently an integer from 0 to 4. The symbols ml,m2 and m3 are
independently an integer
from 1 to 2. The symbols vi, v2 and v3 are independently an integer from 1 to
2.
[0023] In one aspect, a method of increasing global immune activation in a
subject in need
thereof is provided. The method includes administering to the subject a
therapeutically effective
amount of an adenosine-A2A (A2A) receptor antagonist, wherein the A2A receptor
antagonist is
a compound of formula:
RI
R3 (I),
[0024] In formula (I), RI is independently hydrogen, halogen, -CXa3, -CN, -
S02C1, -S0n1R9,
_oNR9Rio, _
NHC=(0)NHNH2, -NHC=(0)NR9R1 , -N(0)mi,-
NR9Rto,
-NH-0-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9Rio, _OR9, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S012R11,
-S0,2NRitRi2, NHNH2, 0NRI IR' 2, NHc_(0)NHNH2, Nix _(0)NRIIR12, _N(0),2,
_NRItRi2, _NH_O_Rii, -C(0)R", -C(0)-OR", -C(0)NRItR12, -OR", ,
SLID stitutea or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CX'3, -CN, -
S02C1, -S0n3R13,
-S0,3NRi3R14, _NHNH2, _0NRi3R14, _NHc=(0)NHNH2, _NHc=(0)NRI3Ri4,_N(0),13,

CA 03009527 2018-06-21
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_NRnR14, -C(0)R'3, _C(0)-0R13, -C(0)NR13R14, _0R'3, =
substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, Rio, Rn, R12, R13 and It¨ 14
are independently hydrogen, halogen,
=0, =S, -CF3,-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
SO4H,
-SO2NH2, -NO2, -NHNH2, -0N112, -NTC=(0)NHNH2, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. Xa, Xb and Xc are independently ¨F, -Cl, -Br, or ¨I. The symbols
ni, nz and n3 are
independently an integer from 0 to 4. The symbols ml, m2 and m3 are
independently an integer
from 1 to 2. The symbols vi, v2 and v3 are independently an integer from 1 to
2.
[0025] In one aspect, a method of treating cancer in a subject in need thereof
is provided. The
method includes administering to the subject a therapeutically effective
amount of an adenosine-
A2A (A2A) receptor antagonist of formula:
_(
N
N/
\\N N H2
______ N
0
0 , wherein the adenosine-A2A (A2A) receptor
antagonist is
administered at 100 mg twice a day (BID).
[0026] In one aspect, a method of treating cancer in a subject in need thereof
is provided. The
method includes administering to the subject a therapeutically effective
amount of an adenosine-
A2A (A2A) receptor antagonist of formula:
11

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PCT/US2016/068459
N
-N
0
0 and
a therapeutically effective amount of atezolizumab.
[0027] In one aspect, a pharmaceutical composition including an A2A receptor
antagonist, a
PD-1 signaling pathway inhibitor and a pharmaceutically acceptable excipient
is provided.
[0028] In one aspect, a pharmaceutical composition including an A2A receptor
antagonist of
formula:
N
\NH2
0
0 and
a pharmaceutically acceptable excipient is provided,
wherein the adenosine-A2A (A2A) receptor antagonist is present at 100 mg.
[0029] In one aspect, a pharmaceutical composition including an adenosine-A2A
(A2A)
receptor antagonist of formula:
12

CA 03009527 2018-06-21
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N
-N
0
0 atezolizumab and a pharmaceutically acceptable
excipient
is provided.
[0030] In one aspect, a method of activating a T cell is provided. The method
includes
contacting the T cell with an A2A receptor antagonist, wherein the adenosine-
A2A (A2A)
receptor antagonist is a compound of formula:
___________ \
N)-2
0
0
[0031] In one aspect, a method of inhibiting A2A receptor activity of a cell
is provided. The
method includes contacting said cell with an A2A receptor antagonist, wherein
the A2A receptor
antagonist is a compound of formula:
13

CA 03009527 2018-06-21
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N
NN
N112
-N
0
[0032] In one aspect, a method of increasing an anti-tumor immune response in
a subject in
need thereof is provided. The method includes administering to the subject a
therapeutically
effective amount of an adenosine-A2A (A2A) receptor antagonist, wherein the
adenosine-A2A
(A2A) receptor antagonist is a compound of formula:
N
NH2
-N
0
0
[0033] In one aspect, a method of of increasing the amount of CD8-positive
cells relative to
the amount of regulatory T cells in a subject in need thereof is provided. The
method includes
administering to the subject a therapeutically effective amount of an
adenosine-A2A (A2A)
14

CA 03009527 2018-06-21
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receptor antagonist, wherein the adenosine-A2A (A2A) receptor antagonist is a
compound of
formula:
N
.,..2
0
0
[0034] In one aspect, a method of decreasing tumor volume in a subject in need
thereof is
provided. The method includes administering to said subject a therapeutically
effective amount
of an adenosine-A2A (A2A) receptor antagonist, wherein the adenosine-A2A (A2A)
receptor
antagonist is a compound of formula:
"
________________________ \
NH2
-N
0
0

CA 03009527 2018-06-21
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[0035] In one aspect, a method of of enhancing anti-tumor immune memory in a
subject in
need thereof is provided. The method includes administering to said subject a
therapeutically
effective amount of an adenosine-A2A (A2A) receptor antagonist, wherein the
adenosine-A2A
(A2A) receptor antagonist is a compound of formula:
N
1/7
N N 112
___________________ N
0
0
[0036] In one aspect, a method of detecting a phosphorylated cAMP response
element-binding
protein (pCREB) in a B-cell or T-cell of a mammalian subject is provided. The
method includes:
(i) obtaining a blood sample from a mammalian subject;
(ii) contacting the blood sample with an adenosine receptor agonist;
(iii) contacting the blood sample with a pCREB detection agent and a blood
cell
detection agent, wherein the blood cell detection agent includes a B-cell
detection
agent or T-cell detection agent, thereby forming a T-cell-detection agent
complex
or a B-cell-detection agent complex; and
(iv) detecting the T-cell detection agent complex or the B-cell detection
complex
thereby detecting the pCREB in a T-cell or B-cell.
[0037] In one aspect, the adenosine receptor agonist includes adenosine, 5'-N-
Ethylcarboxamidoadenosine (NECA), or an analog thereof. In one aspect, the
pCREB detection
agent includes an antibody against pCREB. In one aspect, the B cell detection
agent includes an
16

CA 03009527 2018-06-21
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antibody against CD19 and/or an antibody against CD20. In one aspect, the T
cell detection
agent includes an antibody against CD3, CD4 and/or an antibody against CD8.
[0038] In one aspect, the method further includes contacting the blood sample
with a fixation
agent and cell permeabilizing agent after contacting the blood sample with an
adenonsine
receptor agonist and prior to contacting the blood sample with a pCREB
detection agent. In one
aspect, the method further includes contacting the blood sample with an
apoptotic cell detection
agent. In one aspect, the apoptotic cell detection agent includes an antibody
against cPARP. In
one aspect, the method further includes, prior to obtaining the blood sample,
administering to the
mammalian subject an adenosine receptor antagonist.
[0039] In one aspect, the adenosine receptor antagonist includes an A2a
receptor antagonist or
an A2b receptor antagonist. In one aspect, the method further includes, prior
to obtaining the
blood sample, administering to the mammalian subject an anti-cancer agent. In
one aspect, the
anti-cancer agent includes a PD-Li antagonist. In one aspect, the PD-Ll
antagonist includes
atezolizumab. In one aspect, the method further includes, contacting the blood
sample with a
cell subset detection agent. In one aspect, the cell subset detection agent
includes a naive cell
detection agent, a memory cell detection agent, or an effector cell detection
agent. In one aspect,
the cell subset detection agent includes an antibody against CD27 or an
antibody against
CD45RA. In one aspect, the blood sample is collected from circulating blood.
In one aspect, the
blood sample includes an intratumoral sample.
[0040] In one aspect, a method of treating a subject with cancer is provided.
The method
includes:
(i) obtaining a blood sample from a subject with cancer;
(ii) detecting a level of pCREB induced by an adenosine receptor agonist in
the
sample;
(iii) administering an effective amount of an adenosine receptor antagonist
to the
subject.
[0041] In one aspect, the detecting of the level of pCREB induced in the
sample includes:
(a) contacting the blood sample with an adenosine receptor agonist; and
17

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(b) contacting the blood sample with a pCREB detection agent and a blood cell
detection
agent, wherein the blood cell detection agent includes a B-cell detection
agent or T-cell
detection agent.
[0042] In one aspect, the pCREB detection agent includes an antibody against
pCREB. In one
aspect, the B cell detection agent includes an antibody against CD19 and/or
against CD20._ In
one aspect, the T cell detection agent includes an antibody against CD3, CD4
and/or an antibody
against CD8. In one aspect, detecting the level of pCREB induced in the
subject comprises
measuring a level of pCREB in B cells or T cells prior to the administering of
the effective
amount of an adenosine receptor antagonist to the subject.
[0043] In one aspect, the method further includes: (iv) detecting a level of
pCREB induced in
said sample following said administering of the effective amount of adenosine
receptor
antagonist to said subject. In one aspect, the detecting of the level of pCREB
induce in said
sample comprises measuring a level of pCREB induced in B cells or T cells
following said
administering of the effective amount of adenosine receptor antagonist to said
subject.
[0044] In one aspect, the method includes increasing a dose of an adenosine
receptor
antagonist based on the level of pCREB induced in said B cells or T cells.
[0045] In one aspect a peimeabilized blood cell is provided. The permeabilized
blood cell
includes a pCREB detection agent and a blood cell detection agent, wherein the
blood cell
detection agent includes a B-cell detection agent or T-cell detection agent
and the permeabilized
blood cell includes a permeabilized B-cell or permeabilized T-cell. In one
aspect, the
permeabilized blood cell further includes an apoptotic cell detection agent.
In one aspect, the
apoptotic cell detection agent includes an antibody against cPARP. In one
aspect the
permeabilized blood cell further includes a mature cell detection agent. In
one aspect, the mature
cell detection agent includes antibody against CD27 or an antibody against
CD45RA.
[0046] In one aspect, a container including an adenosine receptor agonist in
combination with
the permeabilized cell as described above is provided.
[0047] In one aspect, a flow cytometer including the permeabilized blood cell
as described
above is provided.
18

84342314
[0047a1 In one aspect, there is provided a use of an adenosine-A2A receptor
antagonist of
folinula:
\
0
0
0
NT 12 or a pharmaceutically
acceptable
salt thereof, to treat colon cancer, lung cancer, triple negative breast
cancer, melanoma, head and
neck cancer, prostate cancer, bladder cancer, renal cell carcinoma, colorectal
cancer, or
lymphoma; wherein the adenosine-A2A receptor antagonist is for administration
in an amount of
about 100 mg twice a day.
[0047b] In one aspect, there is provided a use of an adenosine-A2A receptor
antagonist of
formula:
--N
N
0
0
N
0 N
NH2 or a
pharmaceutically acceptable salt thereof, to increase an anti-tumor immune
response in a subject
having colon cancer, lung cancer, triple negative breast cancer, melanoma,
head and neck cancer,
prostate cancer, bladder cancer, renal cell carcinoma, colorectal cancer, or
lymphoma; wherein
the adenosine-A2A receptor antagonist is for administration in an amount of
about 100 mg twice
a day.
[0047c] In one aspect, there is provided a use of an adenosine-A2A receptor
antagonist of
formula:
18a
Date recue/Date received 2023-04-05

84342314
\ ,N
N ---
/0
0
0
NI-12 or a
pharmaceutically acceptable salt thereof, to decrease tumor volume in a
subject having colon
cancer, lung cancer, triple negative breast cancer, melanoma, head and neck
cancer, prostate
cancer, bladder cancer, renal cell carcinoma, colorectal cancer, or lymphoma;
wherein the
adenosine-A2A receptor antagonist is for administration in an amount of about
100 mg twice a
day.
[0047d] In one aspect, there is provided a use of an adenosine-A2A receptor
antagonist of
fommla:
\"
0
N
0
NH2 or a
pharmaceutically acceptable
salt thereof, to enhance anti-tumor immune memory in a subject having colon
cancer, lung
cancer, triple negative breast cancer, melanoma, head and neck cancer,
prostate cancer, bladder
cancer, renal cell carcinoma, colorectal cancer, or lymphoma; wherein the
adenosine-A2A
receptor antagonist is for administration in an amount of about 100 mg twice a
day.
[0047e] In one aspect, there is provided a use of atezolizumab and an
adenosine-A2A receptor
antagonist of formula:
\
N ----
/0
0
0
NH2
18b
Date recue/Date received 2023-04-05

84342314
or a pharmaceutically acceptable salt thereof to treat cancer; wherein the
cancer is colon cancer,
lung cancer, triple negative breast cancer, melanoma, head and neck cancer,
prostate cancer,
bladder cancer, renal cell carcinoma, colorectal cancer, or lymphoma.
18c
Date recue/Date received 2023-04-05

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BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1: CPI-444 Anti-PD-1 In Early CT26 Model. Therapeutic synergy in
combination with anti-PD-1.
[0049] FIG. 2: CPI-444 Anti-PD-Li In MC38 Model. Combination treatment
inhibits
tumor growth.
[0050] FIG. 3: Efficacy Model: MC38 Colon Cancer. Synergy in combination with
anti-PD-
Ll.
[0051] FIG. 4: Efficacy Model: MC38 Colon Cancer. Skewing toward anti-tumor
immune
response in tumors.
[0052] FIG. 5: Efficacy Model: MC38 Colon Cancer. Skewing toward anti-tumor
immune
response in tumors.
[0053] FIG. 6: CPI-444 Inhibits cAMP Production in T cells
[0054] FIG. 7: CPI-444 Restores IL-2 and IFN-y From Activated T Cells. Primary
human
PBMCs were cultured for 1 hour in the presence of an A2Ar agonist (NECA or
CGS21680, 1
[tm) to stimulate the effects of adenosine on immune cell function. Purified
anti-CD3 and anti-
CD28 monoclonal antibodies (1 ug/ml) were then added to activate T cells for
48 hours. NECA
and CG521680 suppressed release of the Thl cytokines IL-1 and IFNy, mimicking
the
immunosuppressive effects of adenosine signaling. Blockade of A2AR with CPI-
444 (1p.M)
neutralized the immunosuppressive effects of NECA and CGS21680 and restored IL-
2 and IFNy
secretion back to levels observed in the absence of exogenous adenosine
signaling (DMSO
control).
[0055] FIG. 8: CPI-444 Does Not Affect Tumor Cell Proliferation In Vitro
[0056] FIG. 9: CPI-444 Restores pERK In CD4+ T Cells
[0057] FIG. 10: CPI-444 Prevents pCREB Induction in B Cells
[0058] FIG. 11: CPI-444 Inhibits EL4 Tumor Growth In Regional Lymph Nodes
[0059] FIG. 12: CPI-444 Inhibits Growth of MC38 Tumors
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[0060] FIG. 13: CT26 Model: Combo extends long-term survival in 80% mice. Oral

administration of control vehicle (40% solution of hydroxypropyl-beta-
cyclodextrin) or CPI-444
(100 mg/kg) was initiated the same day tumors were engrafted (Day 0).
Treatment continued for
12 days. Half of the mice in the vehicle control group as well as half the
mice in the CPI-444
treatment group received anti-PD-1 mAb (RMP1-14, 100 ug/mouse, i.p.) on days
7, 9, 11, and
13. Administration of anti-PD-1 or CPI-444 resulted in an inhibition of tumor
growth, however,
tumors were not completely eradicated by either treatment. Administration of
CPI-444 in
combination with anti-PD-1 stabilized or eliminated tumors in 8/9 mice,
resulting in improved
overall survival for more than 3 weeks following the last dose of CPI-44 or
anti PD-1 antibody.
[0061] FIG.s 14A and 14B: MC38 Model: CPI-444 eliminates tumors in 30 /s of
mice.
Combo eliminates tumors in 50% of mice. MC 38 mouse colon cancer cells were
engrafted onto
the back of syngeneic C57B1/6 mice. Oral administration of control vehicle or
CPI-444 (100
mg/kg) was initiated the same day tumors were engrafted (Day 0). Treatment
continued for 12
days. Half of the mice in the vehicle control group as well as half the mice
in the CPI-444
treatment group received anti-PD-Li mAb (10F.9G2, 200 ug/mouse, i.p.) on days
7, 10,13, and
16. Administration of anti-PD-Li or CPI-444 resulted in an inhibition of tumor
growth,
however, tumors were not completely eradicated by either treatment. In
contrast, administration
of CPI-444 in combination with anti-PD-Li stabilized or eliminated tumors in
5/10 mice.
[0062] FIG.s 15A and 15B: MC38 Model: CPI-444 eliminated tumors in 30% of
mice.
100% protected from rechallenge. Nine mice that achieved complete tumor growth
inhbition at
the end of the CPI-444 dose response study (FIG. 15A) were monitored for signs
of reoccurence
for an additional 6 weeks. No tumor growth was observed, indicating that the
tumor had been
fully eliminated. These mice were challenged with the new engraftment of MC38
tumor cells.
Modest tumor growth was observed in the first 5 days after rechallenge,
however the tumors
were fully rejected in all 9 mice over the following 15 days (FIG. 15B).
Notably, tumor
elimination occurred in the absence of any additional CPI-444 treatment. These
results clearly
demonstrate that CPI-444 treatment can elicit durable systemic anti-tumor
immune memory.
[0063] FIG.s 16A and 16B: MC38 Model: Established tumors eliminated in 8/9
mice. FIG.
16A: MC38 colon cancer cells were engrafted onto the back of syngeneic C57BL/6
mice. Mice

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were treated with CPI-444 (100 mg/kg), anti-PD-Li (10F.9G2, 200 ug), or
appropriate controls
as indicated. All treatment regimens resulted in an inhbition of tumor growth,
however
therapeutic efficacy was optimal when CPI-444 was administered prior to or
concurrent with
anti-PD-Li (FIG. 16B). The combination of CPI-444 with anti PD-Li was
particularly effective
when initiated on established tumors (Day 7) and resulted in full tumor
elimination in 8/9 mice.
[0064] FIG. 17: MC38 Colon Cancer: Cartoon of Dosing Strategies: Determine
optimal order
of CPI-444 and anti-PD-Li.
[0065] FIG.s 18A and 18B: MC38 Colon Cancer: All treatments started on Day 7
(established tumors). Size of tumor volume (FIG. 18A). Cartoon of dosing
strategies (FIG.
18B).
[0066] FIG.s 19A and 19B: T Cell activation in treated subjects. Whole blood
was collected
on Day 1 of Cycles 1, 2, 4 & 8 for flow analysis. The percentage of CD4 and
CD8 T cells that
stained PD-1+ (FIG. 19A) or CD45RA- (memory/effector cells) (FIG. 19B) is
shown. Each line
represents a single subject across time. CPI-444 treatment increases PD-
1+/CD8+ and memory
cell frequencies as a single agent and in combination cohorts, suggesting
activation of T cell
mediated immunity.
[0067] FIG.s 20A-20C: CPI-444 is associated with changes in T cell repertoire.
Whole blood
was collected on Day 1 of Cycles 1 and 2 and PBMCs were prepared. DNA was
extracted from
PBMCs and sequenced for TCR13 repertoire by Adaptive Biotechnologies.
Expansion of pre-
existing and new T cell clones is observed in response to treatment with
single agent CPI-444
(FIG. 20A). Morisita Index measures T cell repertoir similarity comparing pre-
and post-dose
PBMCs. A Morisita Index of 1 is equal to identity, indicating no longitudinal
change. Morisita
Index distribution in single agent and combination cohorts (FIG. 20B). Graph
showing Morisita
Index by cohort (FIG. 20C). CPI-444 induces robust changes in TCR repertoire
in some patients
treated with single agent CPI-444 and in combination with Tecentriqe. Changes
were driven
predominantly by TCR clone expansion (clonality).
[0068] FIG.s 21A-21C: Efficacy by PD-Li status and prior anti-PD-1 treatment
status. The
disease is stable in anti-PD-Li naive and refractory as well as patient PD-L1+
and PD-Li-
tumors (FIG. 21A and FIG. 21B). Tumor regression in a Nivolumab-refractory
lung cancer
21

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patient (Single agent CPI-444 100mg po BID x28 days/cycle)(FIG. 21C). In FIG.
21C, Patient's
Morisita Index was 0.84 and increased clonality was observed following
treatment.
[0069] FIG.s 22A-22C: Relationship between TCR repertoire and efficacy. TCR
repertoire
changes are similar between patients that are naïve and refractory to prior
anti-PD-1 therapy and
may associate with efficacy (FIG. 22A). FIG. 22B shows the change in tumor
size relative to
baseline plotted against the Morisita Index. FIG. 22C shows the change in
tumor size relative to
baseline plotted against baseline clonality.
[0070] FIG.s 23A-23C: CPI-444 efficacy requires CD8+ T cells. MC38 mouse colon
cancer
cells were engrafted onto the back of syngeneic C57BL/6 mice. Oral
administration of control
vehicle or CPI-444 (100 mg/kg) was initiated 7 days after tumors were
engrafted (Day 0) (FIG.
23C). Treatment continued for more than 9 days (FIG. 23C). Half of the mice in
the vehicle
control group as well as half the mice in the CPI-444 treatment group received
anti-PD-Li mAb
(10F.9G2, 200 ug/mouse, i.p.) on days 7, 10, 13, and 16 (FIG. 23C). 100 ug of
Anti-mCD4
(Clone GKI.5) and/or 500 ug of Anti-mCD8 (Clone 53-6.72) was administered on
day 6. T cell
depletion was verified by flow analysis. FIG.s 23A and 23B show the tumor
volume at different
time points since engraftment for the dosing cohorts. These results suggest
CD8+ T cells are
required for the efficacy of CPI-444 alone or in combination with Anti-PD-Li.
[0071] FIG. 24: A schematic showing the role of CPI-444 in CREB
phosphorylation.
[0072] FIG. 25: A Graph indicating that 5.-N-ethylcarboxamido-adenosine
(NECA),NECA, a
synthetic adenosine analog, activates CREB in whole blood.
[0073] FIG. 26: A schematic showing a pharmacokinetic time course of pCREB
induction in
CPI-444 dosing. The pCREB assay is performed at: Day 1 before dosing; Day 14
with PK time
course. Concentrations used are: 50mg BID,100mg BID, and 200mg QD.
[0074] FIG. 27: A series of graphs charting pCREB induction in B cells in a
subject receiving
200QD CPI-444 for 14 days. Trough refers to pharmacokinetic troughs as seen in
FIG. 26.
[0075] FIG. 28: A series of graphs charting pCREB induction in B cells in a
subject receiving
50mg BID CPI-444 + Atezolizumab for 14 days. Trough refers to pharmacokinetic
troughs as
seen in FIG. 26.
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[0076] FIG. 29: A graph showing CREB phosphorylation in B cells at different
concentrations of NECA prior to adenosine receptor antagonist treatment and
after 14 days of
treatment at trough, 1.5 hour, 3 hour, 5 hour and 8 hour time points for a
subject receiving CPI-
444 alone and a subject receiving combination therapy of CPI-444 and
atezolizumab.'
[0077] FIG. 30: A graph showing inhibition of CREB phosphorylation in B cells
relative to
baseline signaling at different concentrations of NECA after 14 days of
adenosine receptor
antagonist treatment at trough, 1.5 hour, 3 hour, 5 hour and 8 hour time
points for subjects
receiving CPI-444 alone (subject 100301: 200mg QD CPI-444; and subject 100303:
100mg BID
CPI-444) and subjects receiving combination therapy of CPI-444 and
atezolizumab (subject
100302: 50mg BID CPI-444 + atezo; and subject 100402: 50mg BID CPI-444 +
atezo).
[0078] FIG. 31: A graph showing CREB phosphorylation in T cells at different
concentrations of NECA at trough, 1.5 hour, 3 hour, 5 hour and 8 hour time
points for a subject
receiving CPI-444 alone (200QD CPI-444 for 14 days) and a subject receiving
combination
therapy of CPI-444 and atezolizumab (50BID CPI-444 +atezolizumab for 14 days).
[0079] FIG. 32: Phase 1/1B clinical trial design. Step 1 shows the biomarker
objectives to 1)
inform dose selection and schedule using pharamacodynamics assays (pCREB and
immune
activation markers) and 2) explore relationships between efficacy and
biomarkers, e.g., immune
activation in serial peripheral blood and tumor biopsy samples. Step 2 shows
the trial design.
[0080] FIG.s 33A-33B: CPI-444 blocks A2AR in treated subjects. Whole blood was

collected on Day 1 pre-treatment and on Day 14 at pre-dose and post dose at
1.5hr, 3hr, 5.5hr
and 8hr time points. Blood was activated with an adenosine analog (NECA) and
subsequently
stained for intracellular phospho-CREB (pCREB) and cell lineage markers for
flow cytometry.
For each Day 14 time point, the percent inhibition of NECA-induced pCREB is
relative to
baseline. FIG. 33A is a graph showing the inhibition of pCREB relative to
baseline levels over
time. Complete and sustained inhibition was seen in 7 of 7 patients treated
with 100 mg BID
single agent CPI-444. Variable inhibition was seen in 200 mg QD and 50 mg BID
cohorts. FIG.
33B shows the inhibition of pCREB relative to baseline for different
concentrations of plasma
CPI-444. PK/PD analysis supports 100 mg BID as the optimal dose for Step 2
dose expansion
23

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cohorts. The A2AR pathway is completely inhibited at CPI-444 exposures greater
than 2000
ng/mL.
[0081] FIG.s 34A-34D: Graphs showing pCREB percent inhibition in B cells
across the 8 hr
time course of Day 14. Each line represents a single patient and each graph
represents a different
does used in step 1 of the clinical trial. Fig 34A shows pCREB percent
inhibition in B cells in
patients who received 50 mg BID of CPI-444. Fig 34B shows pCREB percent
inhibition in B
cells in patients who received 100 mg BID of CPI-444. Fig 34C shows pCREB
percent
inhibition in B cells in patients who received 200 QD of CPI-444. The majority
of patients in the
100mg BM cohort demonstrate high pCREB inhibition at trough and near complete
inhibition
after taking their morning dose. FIG. 34D is a dot plot showing the change in
percent inhibition
between trough (0 hr) and peak (3 hr). There is little fluctuation from trough
to peak in the 100
mg BID dosing group, making 100 mg BID an appropriate dose for continuous
functional
inhibition. The 50 mg BID is not high enough for sustained inhibition and the
200mg QD dose
achieves high peak levels but is not maintained at trough since CPI-444 is
administered once per
day.
[0082] FIG.s 35A-35B: Percent inhibition of CREB phosphorylation by plasma
levels of CPI-
444. Each dot in FIG. 35A and FIG. 35B represents a single time point from a
single subject.
For plasma levels greater than 2,000 ng/mL near complete inhibition was
observed in B cells
(FIG. 35A) and CD4+ T cells (FIG. 35B).
[0083] FIG. 36: pCREB inhibition is correlated between B cells and CD4+ T
cells. Each dot
represents a single time point for a single subject. The x-axis shows pCREB
percent inhibition
in B cells and the y-axis shows pCREB percent inhibition in CD4+ Tcells. There
is a strong
correlation between inhibition in B cells and CD4+ T cells.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
[0084] While various embodiments and aspects of the present invention are
shown and
described herein, it will be obvious to those skilled in the art that such
embodiments and aspects
are provided by way of example only. Numerous variations, changes, and
substitutions will now
occur to those skilled in the art without departing from the invention. It
should be understood
24

84342314
that various alternatives to the embodiments of the invention described herein
may be employed
in practicing the invention.
[0005] The section headings used herein are for organizational purposes only
and are not to be
construed as limiting the subject matter described.
[0006] The abbreviations used herein have their conventional meaning within
the chemical and
biological arts. The chemical structures and formulae set forth herein are
constructed according
to the standard rules of chemical valency known in the chemical arts.
[0007] Where substituent groups are specified by their conventional chemical
formulae,
written from left to right, they equally encompass the chemically identical
substituents that
would result from writing the structure from right to left, e.g., ¨CH20- is
equivalent to -OCH2-.
[0008] The term "alkyl," by itself or as part of another substituent, means,
unless otherwise
stated, a straight (i.e., unbranched) or branched non-cyclic carbon chain (or
carbon), or
combination thereof, which may be fully saturated, mono- or polyunsaturated
and can include di-
and multivalent radicals, having the number of carbon atoms designated (i.e.,
Ci-Cio means one
to ten carbons). Examples of saturated hydrocarbon radicals include, but are
not limited to,
groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,
sec-butyl,
(cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-
heptyl, n-octyl,
and the like. An unsaturated alkyl group is one having one or more double
bonds or triple bonds.
Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-
propenyl, crotyl, 2-
isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-
butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached
to the remainder
of the molecule via an oxygen linker (-0-). An alkyl moiety may be an alkenyl
moiety. An
alkyl moiety may be an alkynyl moiety. An alkyl moiety may be fully saturated.
An alkenyl
may include more than one double bond and/or one or more triple bonds in
addition to the one or
more double bonds. An alkynyl may include more than one triple bond and/or one
or more
double bonds in addition to the one or more triple bonds.
Date recue/Date received 2023-04-05

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[0089] The term "alkylene," by itself or as part of another substituent,
means, unless otherwise
stated, a divalent radical derived from an alkyl, as exemplified, but not
limited by, ¨
CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24
carbon atoms,
with those groups having 10 or fewer carbon atoms being preferred in the
present invention. A
"lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group,
generally having
eight or fewer carbon atoms. The term "alkenylene," by itself or as part of
another substituent,
means, unless otherwise stated, a divalent radical derived from an alkene.
[0090] The term "heteroalkyl," by itself or in combination with another term,
means, unless
otherwise stated, a stable straight or branched non-cyclic chain, or
combinations thereof,
including at least one carbon atom and at least one heteroatom (e.g. 0, N, P,
Si, and S), and
wherein the nitrogen and sulfur atoms may optionally be oxidized, and the
nitrogen heteroatom
may optionally be quaternized. The heteroatom(s) (e.g. 0, N, P, S, and Si) may
be placed at any
interior position of the heteroalkyl group or at the position at which the
alkyl group is attached to
the remainder of the molecule. Examples include, but are not limited to: -CH2-
CH2-0-CH3,
-CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(0)-CH3,
-CH2-CH2-S(0)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-
CH3,
-0-CH3, -0-CH2-CH3, and -CN. Up to two or three heteroatoms may be
consecutive, such as,
for example, -CH2-NH-OCH3 and ¨CH2-0-Si(CH3)3. A heteroalkyl moiety may
include one
heteroatom (e.g., 0, N, S, Si, or P). A heteroalkyl moiety may include two
optionally different
heteroatoms (e.g., 0, N, S, Si, or P). A heteroalkyl moiety may include three
optionally different
heteroatoms (e.g., 0, N, S, Si, or P). A heteroalkyl moiety may include four
optionally different
heteroatoms (e.g., 0, N, S, Si, or P). A heteroalkyl moiety may include five
optionally different
heteroatoms (e.g., 0, N, S, Si, or P). A heteroalkyl moiety may include up to
8 optionally
different heteroatoms (e.g., 0, N, 5, Si, or P). The term "heteroalkenyl," by
itself or in
combination with another term, means, unless otherwise stated, a heteroalkyl
including at least
one double bond. A heteroalkenyl may optionally include more than one double
bond and/or one
or more triple bonds in additional to the one or more double bonds. The term
"heteroalkynyl,"
by itself or in combination with another term, means, unless otherwise stated,
a heteroalkyl
including at least one triple bond. A heteroalkynyl may optionally include
more than one triple
bond and/or one or more double bonds in additional to the one or more triple
bonds.
26

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[0091] Similarly, the term "heteroalkylene," by itself or as part of another
substituent, means,
unless otherwise stated, a divalent radical derived from heteroalkyl, as
exemplified, but not
limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene
groups,
heteroatoms can also occupy either or both of the chain termini (e.g.,
alkyleneoxy,
alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further,
for alkylene and
heteroalkylene linking groups, no orientation of the linking group is implied
by the direction in
which the formula of the linking group is written. For example, the formula -
C(0)2R1- represents
both -C(0)2R'- and -R'C(0)2-. As described above, heteroalkyl groups, as used
herein, include
those groups that are attached to the remainder of the molecule through a
heteroatom, such
as -C(0)R', -C(0)NR', -NR'R", -OR', -SR', and/or -SO2R'. Where "heteroalkyl"
is recited,
followed by recitations of specific heteroalkyl groups, such as -NR'R" or the
like, it will be
understood that the terms heteroalkyl and -NR'R" are not redundant or mutually
exclusive.
Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the
term "heteroalkyl"
should not be interpreted herein as excluding specific heteroalkyl groups,
such as -NR'R" or the
like.
[0092] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in
combination with
other terms, mean, unless otherwise stated, non-aromatic cyclic versions of
"alkyl" and
"heteroalkyl," respectively, wherein the carbons making up the ring or rings
do not necessarily
need to be bonded to a hydrogen due to all carbon valencies participating in
bonds with non-
hydrogen atoms. Additionally, for heterocycloalkyl, a heteroatom can occupy
the position at
which the heterocycle is attached to the remainder of the molecule. Examples
of cycloalkyl
include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, 1-cyclohexenyl,
3-cyclohexenyl, cycloheptyl, 3-hydroxy-cyclobut-3-eny1-1,2, di one, 1H- 1,2,4-
triazoly1-5(4H)-
one, 4H-1,2,4-triazolyl, and the like. Examples of heterocycloalkyl include,
but are not limited
to, 1-(1,2,5,6-tetrahydropyridy1), 1-piperidinyl, 2-piperidinyl, 3-
piperidinyl, 4-morpholinyl, 3-
morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,
tetrahydrothien-3-
yl, 1-piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a
"heterocycloalkylene,"
alone or as part of another substituent, means a divalent radical derived from
a cycloalkyl and
heterocycloalkyl, respectively. A heterocycloalkyl moiety may include one ring
heteroatom
(e.g., 0, N, S, Si, or P). A heterocycloalkyl moiety may include two
optionally different ring
27

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heteroatoms (e.g., 0, N, S, Si, or P). A heterocycloalkyl moiety may include
three optionally
different ring heteroatoms (e.g., 0, N, S, Si, or P). A heterocycloalkyl
moiety may include four
optionally different ring heteroatoms (e.g., 0, N, S, Si, or P). A
heterocycloalkyl moiety may
include five optionally different ring heteroatoms (e.g., 0, N, S, Si, or P).
A heterocycloalkyl
moiety may include up to 8 optionally different ring heteroatoms (e.g., 0, N,
S, Si, or P).
[0093] The terms "halo" or "halogen," by themselves or as part of another
substituent, mean,
unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
Additionally, terms such
as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For
example, the term
"halo(CI-C4)alkyl" includes, but is not limited to, fluoromethyl,
difluoromethyl, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
[0094] The term "acyl" means, unless otherwise stated, -C(0)R where R is a
substituted or
unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted
heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl.
[0095] The term "aryl" means, unless otherwise stated, a polyunsaturated,
aromatic,
hydrocarbon substituent, which can be a single ring or multiple rings
(preferably from 1 to 3
rings) that are fused together (i.e., a fused ring aryl) or linked covalently.
A fused ring aryl refers
to multiple rings fused together wherein at least one of the fused rings is an
aryl ring. The term
"heteroaryl" refers to aryl groups (or rings) that contain at least one
heteroatom such as N, 0, or
S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the
nitrogen atom(s) are
optionally quaternized. Thus, the term "heteroaryl" includes fused ring
heteroaryl groups (i.e.,
multiple rings fused together wherein at least one of the fused rings is a
heteroaromatic ring). A
5,6-fused ring heteroarylene refers to two rings fused together, wherein one
ring has 5 members
and the other ring has 6 members, and wherein at least one ring is a
heteroaryl ring. Likewise, a
6,6-fused ring heteroarylene refers to two rings fused together, wherein one
ring has 6 members
and the other ring has 6 members, and wherein at least one ring is a
heteroaryl ring. And a 6,5-
fused ring heteroarylene refers to two rings fused together, wherein one ring
has 6 members and
the other ring has 5 members, and wherein at least one ring is a heteroaryl
ring. A heteroaryl
group can be attached to the remainder of the molecule through a carbon or
heteroatom. Non-
28

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limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-
naphthyl, 4-
biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-
imidazolyl, pyrazinyl,
2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-
isoxazolyl, 5-
isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-
thienyl, 3-thienyl, 2-pyridyl, 3-
pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-
benzimidazolyl, 5-
indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-
quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring systems are
selected from the
group of acceptable substituents described below. An "arylene" and a
"heteroarylene," alone or
as part of another sub stituent, mean a divalent radical derived from an aryl
and heteroaryl,
respectively. Non-limiting examples of aryl and heteroaryl groups include
pyridinyl,
pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl,
benzodioxolyl,
benzodioxanyl, thianaphthanyl, pyrrolopyridinyl, indazolyl, quinolinyl,
quinoxalinyl,
pyridopyrazinyl, quinazolinonyl, benzoisoxazolyl, imidazopyridinyl,
benzofuranyl, benzothienyl,
benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl,
pyrazinyl,
oxazolyl, isoxazolyl, thiazolyl, furylthienyl, pyridyl, pyrimidyl,
benzothiazolyl, purinyl,
benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl,
triazolyl, tetrazolyl,
benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl, pyrrolopyrimidinyl,
benzotriazolyl,
benzoxazolyl, or quinolyl. The examples above may be substituted or
unsubstituted and divalent
radicals of each heteroaryl example above are non-limiting examples of
heteroarylene. A
heteroaryl moiety may include one ring heteroatom (e.g., 0, N, or S). A
heteroaryl moiety may
include two optionally different ring heteroatoms (e.g., 0, N, or S). A
heteroaryl moiety may
include three optionally different ring heteroatoms (e.g., 0, N, or S). A
heteroaryl moiety may
include four optionally different ring heteroatoms (e.g., 0, N, or S). A
heteroaryl moiety may
include five optionally different ring heteroatoms (e.g., 0, N, or S). An aryl
moiety may have a
single ring. An aryl moiety may have two optionally different rings. An aryl
moiety may have
three optionally different rings. An aryl moiety may have four optionally
different rings. A
heteroaryl moiety may have one ring. A heteroaryl moiety may have two
optionally different
rings. A heteroaryl moiety may have three optionally different rings. A
heteroaryl moiety may
have four optionally different rings. A heteroaryl moiety may have five
optionally different
rings.
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[0096] A fused ring heterocyloalkyl-aryl is an aryl fused to a
heterocycloalkyl. A fused ring
heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A
fused ring
heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A
fused ring
heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another
heterocycloalkyl. Fused
ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring
heterocycloalkyl-
cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each
independently be
unsubstituted or substituted with one or more of the substitutents described
herein.
[0097] The term "oxo," as used herein, means an oxygen that is double bonded
to a carbon
atom.
[0098] The term "alkylsulfonyl," as used herein, means a moiety having the
formula -S(02)-W,
where R' is a substituted or unsubstituted alkyl group as defined above. R'
may have a specified
number of carbons (e.g., "C1-C4 alkylsulfonyl").
[0099] Each of the above terms (e.g., "alkyl", "heteroalkyl", "cycloalkyl",
"heterocycloalkyl",
"aryl", and "heteroaryl") includes both substituted and unsubstituted forms of
the indicated
radical. Preferred substituents for each type of radical are provided below.
[0100] Substituents for the alkyl and heteroalkyl radicals (including those
groups often
referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl,
cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of
a variety of
groups selected from, but not limited to, -OR', =0, =NR', =N-OR', -NR'R", -SW,
-halogen, -SiR'R"R", -0C(0)R', -C(0)R', -CO2R', -CONR'R", -0C(0)NR'R", -
NR"C(0)R',
-NR'-C(0)NR"R", -NR"C(0)2RI, -NR-C(NRIR"R'")=NR"", -NR-C(NR'R")=NR'", -S(0)R',
-S(0)2R, -S(0)2NR'R", -NRSO2R', ¨NR'NR"R", ¨0NR'R", ¨NR'C=(0)NR"NR"'R",
-CN, -NO2, in a number ranging from zero to (2m'+1), where m is the total
number of carbon
atoms in such radical. R, R', R", R'", and R" each preferably independently
refer to hydrogen,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl
substituted with 1-3
halogens), substituted or unsubstituted heteroaryl, substituted or
unsubstituted alkyl, alkoxy, or
thioalkoxy groups, or arylalkyl groups. When a compound of the invention
includes more than
one R group, for example, each of the R groups is independently selected as
are each R', R", R'",

CA 03009527 2018-06-21
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and R" group when more than one of these groups is present. When R' and R" are
attached to
the same nitrogen atom, they can be combined with the nitrogen atom to form a
4-, 5-, 6-, or 7-
membered ring. For example, -NR'R" includes, but is not limited to, 1-
pyrrolidinyl and 4-
morpholinyl. From the above discussion of substituents, one of skill in the
art will understand
that the term "alkyl" is meant to include groups including carbon atoms bound
to groups other
than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl
(e.g., -C(0)CH3, -C(0)CF3, -C(0)CH2OCH3, and the like).
[0101] Similar to the substituents described for the alkyl radical,
substituents for the aryl and
heteroaryl groups are varied and are selected from, for example: -OR', -NR'R",
-SR',
-halogen, -SiR'R"R", -0C(0)R', -C(0)R', -CO2R', -CONR'R", -0C(0)NR'R", -
NR"C(0)R',
-NR'-C(0)NR"R'", -NR"C(0)2R', -NR-C(NR'R"R")=NR", -NR-C(NR'R")=NR", -S(0)R',
-S(0)2R', -S(0)2NR'R", -NRSO2W, ¨NR'NR"R", ¨0NR'R", ¨NR'C=(0)NR"NR"R",
-CN, -NO2, -R', -N3, -CH(Ph)2, fluoro(CI-C4)alkoxy, and fluoro(Ci-C4)alkyl, in
a number
ranging from zero to the total number of open valences on the aromatic ring
system; and where
R', R", R'", and R" are preferably independently selected from hydrogen,
substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, and
substituted or unsubstituted heteroaryl. When a compound of the invention
includes more than
one R group, for example, each of the R groups is independently selected as
are each R', R",
and R" groups when more than one of these groups is present.
[0102] Two or more substituents may optionally be joined to form aryl,
heteroaryl, cycloalkyl,
or heterocycloalkyl groups. Such so-called ring-forming substituents are
typically, though not
necessarily, found attached to a cyclic base structure. In one embodiment, the
ring-forming
substituents are attached to adjacent members of the base structure. For
example, two ring-
forming substituents attached to adjacent members of a cyclic base structure
create a fused ring
structure. In another embodiment, the ring-forming substituents are attached
to a single member
of the base structure. For example, two ring-forming substituents attached to
a single member of
a cyclic base structure create a spirocyclic structure. In yet another
embodiment, the ring-
forming substituents are attached to non-adjacent members of the base
structure.
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[0103] Two of the substituents on adjacent atoms of the aryl or heteroaryl
ring may optionally
form a ring of the formula -T-C(0)-(CRR')q-U-, wherein T and U are
independently -NR-, -0-, -CRR'-, or a single bond, and q is an integer of from
0 to 3.
Alternatively, two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may
optionally be replaced with a substituent of the formula -A-(CH2),-B-, wherein
A and B are
independently -CRW-, -0-, -S-, -S(0) -, -S(0)2-, -S(0)2NR'-, or a single
bond, and r is an
integer of from 1 to 4. One of the single bonds of the new ring so formed may
optionally be
replaced with a double bond. Alternatively, two of the substituents on
adjacent atoms of the aryl
or heteroaryl ring may optionally be replaced with a substituent of the
formula
-(CRR'),-X'- (C"R"R")d-, where s and d are independently integers of from 0 to
3, and X'
is -0-, -NR'-, -S-, -S(0)-, -S(0)2-, or -S(0)2NR'-. The substituents R, R',
R", and R" are
preferably independently selected from hydrogen, substituted or unsubstituted
alkyl, substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, and substituted or
unsubstituted heteroaryl.
[0104] As used herein, the terms "heteroatom" or "ring heteroatom" are meant
to include,
oxygen (0), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
101051 A "substituent group," as used herein, means a group selected from the
following
moieties:
(A) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -S03H, -
SO4H,
-SO2NH2, -NHNH2, -0NH2, -NHC=(0)NHNH2, -NHC=(0) NH2, -NHSO2H,
-NHC= (0)H, -NHC(0)-0H, -NHOH, -0CF3, -OCHF2, unsubstituted alkyl,
unsubstituted
heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,
unsubstituted aryl,
unsubstituted heteroaryl, and
(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,
substituted with at least
one substituent selected from:
(i) oxo, halogen, -CF3, -CN, -OH, -COOH, -CONH2, -NO2, -SH, -S03H, -SO4H,

-SO2NH2, -NHNH2, -ONH2, -NHC-(0)NHNH2, -NHC-(0) NH2, -NHSO2H,
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-NHC= (0)H, -NIC(0)-OH, -NHOH, -0CF3, -OCHF2, unsubstituted alkyl,
unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl,
unsubstituted aryl, unsubstituted heteroaryl, and
(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,
substituted with at
least one substituent selected from:
(a) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -S03H,
-SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, -NHC=(0) NH2,
-NHSO2H, -NHC= (0)H, -NFIC(0)-0H, -NHOH, -0CF3, -OCHF2, unsubstituted
alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,
substituted with
at least one substituent selected from: oxo, halogen, -CF3, -CN, -OH, -NH2,
-COOH, -CONH2, -NO2, -SH, -SO 3H, -SO4H, -SO2NH2, -NTNH2, -ONH2,
-NHC=(0)NHNH2, -NHC=(0) NH2, -NHSO2H, -NHC= (0)H,
-N1-1C(0)-0H, -NHOH, -0CF3, -OCHF2, unsubstituted alkyl, unsubstituted
heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,
unsubstituted
aryl, unsubstituted heteroaryl.
[0106] A "size-limited substituent" or" size-limited substituent group," as
used herein, means
a group selected from all of the substituents described above for a
"substituent group," wherein
each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-
C20 alkyl, each
substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2
to 20 membered
heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or
unsubstituted C 3-C 8
cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a
substituted or unsubstituted 3
to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a
substituted or
unsubstituted C6-Clo aryl, and each substituted or unsubstituted heteroaryl is
a substituted or
unsubstituted 5 to 10 membered heteroaryl.
[0107] A "lower substituent" or" lower substituent group," as used herein,
means a group
selected from all of the substituents described above for a "substituent
group," wherein each
substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8
alkyl, each substituted or
unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered
heteroalkyl, each
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substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-
C7 cycloalkyl, each
substituted or unsubstituted heterocycloalkyl is a substituted or
unsubstituted 3 to 7 membered
heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or
unsubstituted C6-C10
aryl, and each substituted or unsubstituted heteroaryl is a substituted or
unsubstituted 5 to 9
membered heteroaryl.
[0108] In some embodiments, each substituted group described in the compounds
herein is
substituted with at least one substituent group. More specifically, in some
embodiments, each
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl,
substituted aryl, substituted heteroaryl, substituted alkylene, substituted
heteroalkylene,
substituted cycloalkylene, substituted heterocycloalkylene, substituted
arylene, and/or substituted
heteroarylene described in the compounds herein are substituted with at least
one substituent
group. In other embodiments, at least one or all of these groups are
substituted with at least one
size-limited substituent group. In other embodiments, at least one or all of
these groups are
substituted with at least one lower substituent group.
[0109] In other embodiments of the compounds herein, each substituted or
unsubstituted alkyl
may be a substituted or unsubstituted Ci-C20 alkyl, each substituted or
unsubstituted heteroalkyl
is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each
substituted or unsubstituted
cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each
substituted or unsubstituted
heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered
heterocycloalkyl, each
substituted or unsubstituted aryl is a substituted or unsubstituted C6-C to
aryl, and/or each
substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to
10 membered
heteroaryl. In some embodiments of the compounds herein, each substituted or
unsubstituted
alkylene is a substituted or unsubstituted C1-C20 alkylene, each substituted
or unsubstituted
heteroalkylene is a substituted or unsubstituted 2 to 20 membered
heteroalkylene, each
substituted or unsubstituted cycloalkylene is a substituted or unsubstituted
C3-C8 cycloalkylene,
each substituted or unsubstituted heterocycloalkylene is a substituted or
unsubstituted 3 to 8
membered heterocycloalkylene, each substituted or unsubstituted arylene is a
substituted or
unsubstituted C6-C10 arylene, and/or each substituted or unsubstituted
heteroarylene is a
substituted or unsubstituted 5 to 10 membered heteroarylene.
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101101 In some embodiments, each substituted or unsubstituted alkyl is a
substituted or
unsubstituted CI-Cs alkyl, each substituted or unsubstituted heteroalkyl is a
substituted or
unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted
cycloalkyl is a
substituted or unsubstituted C3-C7 cycloalkyl, each substituted or
unsubstituted heterocycloalkyl
is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each
substituted or
unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and/or each
substituted or
unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered
heteroaryl. In some
embodiments, each substituted or unsubstituted alkylene is a substituted or
unsubstituted CI-Cs
alkylene, each substituted or unsubstituted heteroalkylene is a substituted or
unsubstituted 2 to 8
membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a
substituted or
unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted
heterocycloalkylene is a
substituted or unsubstituted 3 to 7 membered heterocycloalkylene, each
substituted or
unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene, and/or
each substituted or
unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered
heteroarylene. In
some embodiments, the compound is a chemical species set forth in the Examples
section,
figures, or tables below.
101111 The term "pharmaceutically acceptable salts" is meant to include salts
of the active
compounds that are prepared with relatively nontoxic acids or bases, depending
on the particular
substituents found on the compounds described herein. When compounds of the
present
invention contain relatively acidic functionalities, base addition salts can
be obtained by
contacting the neutral form of such compounds with a sufficient amount of the
desired base,
either neat or in a suitable inert solvent. Examples of pharmaceutically
acceptable base addition
salts include sodium, potassium, calcium, ammonium, organic amino, or
magnesium salt, or a
similar salt. When compounds of the present invention contain relatively basic
functionalities,
acid addition salts can be obtained by contacting the neutral form of such
compounds with a
sufficient amount of the desired acid, either neat or in a suitable inert
solvent. Examples of
pharmaceutically acceptable acid addition salts include those derived from
inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,

monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric,
hydri odic, or
phosphorous acids and the like, as well as the salts derived from relatively
nontoxic organic acids

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like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,
suberic, fumaric, lactic,
mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the
like. Also included are salts of amino acids such as arginate and the like,
and salts of organic
acids like glucuronic or galactunoric acids and the like (see, e.g., Berge et
al., Journal of
Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds of the
present invention
contain both basic and acidic functionalities that allow the compounds to be
converted into either
base or acid addition salts. Other pharmaceutically acceptable carriers known
to those of skill in
the art are suitable for the present invention. Salts tend to be more soluble
in aqueous or other
protonic solvents than are the corresponding free base forms. In other cases,
the preparation may
be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7%
mannitol at a pH
range of 4.5 to 5.5, that is combined with buffer prior to use.
[0112] Thus, the compounds of the present invention may exist as salts, such
as with
pharmaceutically acceptable acids. The present invention includes such salts.
Examples of such
salts include hydrochlorides, hydrobromides, sulfates, methanesulfonates,
nitrates, maleates,
acetates, citrates, fumarates, tartrates (e.g., ( )-tartrates, (-)-tartrates,
or mixtures thereof
including racemic mixtures), succinates, benzoates, and salts with amino acids
such as glutamic
acid. These salts may be prepared by methods known to those skilled in the
art.
[0113] The neutral forms of the compounds are preferably regenerated by
contacting the salt
with a base or acid and isolating the parent compound in the conventional
manner. The parent
form of the compound differs from the various salt forms in certain physical
properties, such as
solubility in polar solvents.
[0114] Provided herein are agents (e.g. compounds, drugs, therapeutic agents)
that may be in a
prodrug form. Prodrugs of the compounds described herein are those compounds
that readily
undergo chemical changes under select physiological conditions to provide the
final agents (e.g.
compounds, drugs, therapeutic agents). Additionally, prodrugs can be converted
to agents (e.g.
compounds, drugs, therapeutic agents) by chemical or biochemical methods in an
ex vivo
environment. Prodrugs described herein include compounds that readily undergo
chemical
changes under select physiological conditions to provide agents (e.g.
compounds, drugs,
therapeutic agents) to a biological system (e.g. in a subject).
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[0115] Certain compounds of the present invention can exist in unsolvated
forms as well as
solvated forms, including hydrated forms. In general, the solvated forms are
equivalent to
unsolvated forms and are encompassed within the scope of the present
invention. Certain
compounds of the present invention may exist in multiple crystalline or
amorphous forms. In
general, all physical forms are equivalent for the uses contemplated by the
present invention and
are intended to be within the scope of the present invention.
[0116] As used herein, the term "salt" refers to acid or base salts of the
compounds used in the
methods of the present invention. Illustrative examples of acceptable salts
are mineral acid
(hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts,
organic acid (acetic
acid, propionic acid, glutamic acid, citric acid and the like) salts,
quaternary ammonium (methyl
iodide, ethyl iodide, and the like) salts.
[0117] Certain compounds of the present invention possess asymmetric carbon
atoms (optical
or chiral centers) or double bonds; the enantiomers, racemates, diastereomers,
tautomers,
geometric isomers, stereoisometric forms that may be defined, in terms of
absolute
stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and
individual isomers are
encompassed within the scope of the present invention. The compounds of the
present invention
do not include those which are known in art to be too unstable to synthesize
and/or isolate. The
present invention is meant to include compounds in racemic and optically pure
forms. Optically
active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral
synthons or chiral
reagents, or resolved using conventional techniques. When the compounds
described herein
contain olefinic bonds or other centers of geometric asymmetry, and unless
specified otherwise,
it is intended that the compounds include both E and Z geometric isomers.
[0118] As used herein, the term "isomers" refers to compounds having the same
number and
kind of atoms, and hence the same molecular weight, but differing in respect
to the structural
arrangement or configuration of the atoms.
[0119] The term "tautomer," as used herein, refers to one of two or more
structural isomers
which exist in equilibrium and which are readily converted from one isomeric
form to another.
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[0120] It will be apparent to one skilled in the art that certain compounds of
this invention may
exist in tautomeric forms, all such tautomeric forms of the compounds being
within the scope of
the invention.
[0121] Unless otherwise stated, structures depicted herein are also meant to
include all
stereochemical forms of the structure; i.e., the R and S configurations for
each asymmetric
center. Therefore, single stereochemical isomers as well as enantiomeric and
diastereomeric
mixtures of the present compounds are within the scope of the invention.
[0122] Unless otherwise stated, structures depicted herein are also meant to
include
compounds which differ only in the presence of one or more isotopically
enriched atoms. For
example, compounds having the present structures except for the replacement of
a hydrogen by a
deuterium or tritium, or the replacement of a carbon by 13C- or 14C-enriched
carbon are within
the scope of this invention.
[0123] The compounds of the present invention may also contain unnatural
proportions of
atomic isotopes at one or more of the atoms that constitute such compounds.
For example, the
compounds may be radiolabeled with radioactive isotopes, such as for example
tritium (3H),
iodine-125 (1251), or carbon-14 (14C). All isotopic variations of the
compounds of the present
invention, whether radioactive or not, are encompassed within the scope of the
present invention.
[0124] The symbol denotes the point of attachment of a chemical moiety to
the
remainder of a molecule or chemical formula.
[0125] In embodiments, a compound as described herein may include multiple
instances of R2
and/or other variables. In such embodiments, each variable may optional be
different and be
appropriately labeled to distinguish each group for greater clarity. For
example, where each R2
..
is different, they may be referred to, for example, as R2',

R22,

R23,

and/or R2.4 respectively,
wherein the definition of R2 is assumed by R2.1, R2.2, R2.3,
and/or R2-4. The variables used within
a definition of R2 and/or other variables that appear at multiple instances
and are different may
similarly be appropriately labeled to distinguish each group for greater
clarity. In some
embodiments, the compound is a compound described herein (e.g., in an aspect,
embodiment,
example, claim, table, scheme, drawing, or figure).
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[0126] The terms "a" or "an," as used in herein means one or more. In
addition, the phrase
"substituted with a[n]," as used herein, means the specified group may be
substituted with one or
more of any or all of the named substituents. For example, where a group, such
as an alkyl or
heteroaryl group, is "substituted with an unsubstituted C1-C20 alkyl, or
unsubstituted 2 to 20
membered heteroalkyl," the group may contain one or more unsubstituted C1-C20
alkyls, and/or
one or more unsubstituted 2 to 20 membered heteroalkyls.
[0127] Where a moiety is substituted with an R substituent, the group may be
referred to as
"R-substituted." Where a moiety is R-substituted, the moiety is substituted
with at least one R
substituent and each R substituent is optionally different. For example, where
a moiety herein is
R12-substituted or unsubstituted alkyl, a plurality of R1-2 substituents may
be attached to the alkyl
moiety wherein each R12 substituent is optionally different. Where an R-
substituted moiety is
substituted with a plurality R substituents, each of the R-substituents may be
differentiated herein
using a prime symbol 0 such as R', R", etc. For example, where a moiety is R12-
substituted or
unsubstituted alkyl, and the moiety is substituted with a plurality of Ril
substituents, the plurality
of R12 substituents may be differentiated as R12', R1-2", R12"1, etc. In
embodiments, the plurality of
R substituents is 3, In embodiments, the plurality of R substituents is 2.
[0128] In embodiments, a compound as described herein may include multiple
instances of RI,
R2, R3, R4, R5, R6, R7, R9, R10, RH, R12, R13, - 14
K and/or other variables. In such embodiments,
each variable may optional be different and be appropriately labeled to
distinguish each group
for greater clarity. For example, where each R1, R2, R3, R4, R5, R6, R7, R9,
R10, R11, R12, R13,
and/or le'', is different, they may be referred to, for example, as R1.1,
Ri.2, R1.3, R1.4, R11, R2.2,
R2.3, R24, R3.1, R3.2, R3.3, R34, R4.1, R4.2, R43, R4.4, R5.1, R52, R5.3, RSA,
R6.1, R6.2, R6.3, R6.47 R7.1,
R7.2, R7.3, R7.4, R9.1, R9.2, R9.3, R9.4, R10.1, R10.2, R10.3, R10.4, R11.1,
R11.2, R11.3, R11.4, R12.1, R122,
R113, R12.4, R13.1, R13.2, R13.3, R13.4, R14.1, R14.2, R14.3, and/or RI",
respectively, wherein the
definition of RI- is assumed by Rid, Ri.2, R1.3,
and/or R", the definition of R2 is assumed by RI',
R2.2, R2.3, and/or R2.4, the definition of R3 is assumed by R3.1, R3.2, R33,
and/or R34, the definition
of R4 is assumed by R4.1, R4.2, R4.3, and/or R4A, the definition of R5 is
assumed by R5-', R5.2, R53,
and/or R54, the definition of R6 is assumed by R6.1, R6.2, R6.3, and/or R6.4,
the definition of R7 is
.1, R7.2, R7.3,
assumed by R7 and/or R7-4, the definition of IR9 is assumed by R9-1,
R9.2, R9-3, and/or
R9.4, the definition of RH' is assumed by R10.1, R10.2, R10.3, and/or R10.4,
the definition of is
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assumed by R".1, R11.2, R11.3,
and/or R11.4, the definition of R12 is assumed by R12.1, R12,2, R123,
and/or R12.4, the definition of R13 is assumed by R13.1, R13.2, R13.3, and/or
RI-34, the definition of
R14 is assumed by R14.1, R14.2, R14.3, and/or RNA. The variables used within a
definition of R1,
R2, R3, R4, R5, R6, R7, R9, Rif), RH, R12, ¨13
K
and/or RH, and/or other variables that appear at
multiple instances and are different may similarly be appropriately labeled to
distinguish each
group for greater clarity.
101291 Descriptions of compounds of the present invention are limited by
principles of
chemical bonding known to those skilled in the art. Accordingly, where a group
may be
substituted by one or more of a number of substituents, such substitutions are
selected so as to
comply with principles of chemical bonding and to give compounds which are not
inherently
unstable and/or would be known to one of ordinary skill in the art as likely
to be unstable under
ambient conditions, such as aqueous, neutral, and several known physiological
conditions. For
example, a heterocycloalkyl or heteroaryl is attached to the remainder of the
molecule via a ring
heteroatom in compliance with principles of chemical bonding known to those
skilled in the art
thereby avoiding inherently unstable compounds.
101301 Antibodies are large, complex molecules (molecular weight of ¨150,000
or about 1320
amino acids) with intricate internal structure. A natural antibody molecule
contains two identical
pairs of polypeptide chains, each pair having one light chain and one heavy
chain. Each light
chain and heavy chain in turn consists of two regions: a variable ("V") region
involved in
binding the target antigen, and a constant ("C") region that interacts with
other components of
the immune system. The light and heavy chain variable regions come together in
3-dimensional
space to form a variable region that binds the antigen (for example, a
receptor on the surface of a
cell). Within each light or heavy chain variable region, there are three short
segments (averaging
amino acids in length) called the complementarity determining regions
("CDRs"). The six
CDRs in an antibody variable domain (three from the light chain and three from
the heavy chain)
fold up together in 3-dimensional space to form the actual antibody binding
site which docks
onto the target antigen. The position and length of the CDRs have been
precisely defined by
Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S.
Department of Health and
Human Services, 1983, 1987. The part of a variable region not contained in the
CDRs is called
the framework ("FR"), which forms the environment for the CDRs.

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[0131] The term "antibody" is used according to its commonly known meaning in
the art. As
used herein, "antibody" may also refer to the antigen binding fragment thereof
Antibodies exist,
e.g., as intact immunoglobulins or as a number of well-characterized fragments
produced by
digestion with various peptidases. Thus, for example, pepsin digests an
antibody below the
disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab
which itself is a light
chain joined to VH-CHI by a disulfide bond. The F(ab)'2 may be reduced under
mild conditions
to break the disulfide linkage in the hinge region, thereby converting the
F(ab)'2 dimer into an
Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge
region (see
Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody
fragments are
defined in terms of the digestion of an intact antibody, one of skill will
appreciate that such
fragments may be synthesized de novo either chemically or by using recombinant
DNA
methodology. Thus, the teiin antibody, as used herein, also includes antibody
fragments either
produced by the modification of whole antibodies, or those synthesized de novo
using
recombinant DNA methodologies (e.g., single chain Fv) or those identified
using phage display
libraries (see, e.g., McCafferty etal., Nature 348:552-554 (1990)).
[0132] A single-chain variable fragment (scFv) is typically a fusion protein
of the variable
regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected
with a short
linker peptide of 10 to about 25 amino acids. The linker may usually be rich
in glycine for
flexibility, as well as serine or threonine for solubility. The linker can
either connect the N-
teiminus of the VII with the C-terminus of the VL, or vice versa.
[0133] For preparation of monoclonal or polyclonal antibodies, any technique
known in the art
can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor
et al.,
Immunology Today 4:72 (1983); Cole etal., pp. 77-96 in Monoclonal Antibodies
and Cancer
Therapy (1985)). "Monoclonal" antibodies (mAb) refer to antibodies derived
from a single
clone. Techniques for the production of single chain antibodies (U.S. Pat. No.
4,946,778) can be
adapted to produce antibodies to polypeptides of this invention. Also,
transgenic mice, or other
organisms such as other mammals, may be used to express humanized antibodies.
Alternatively,
phage display technology can be used to identify antibodies and heteromeric
Fab fragments that
specifically bind to selected antigens (see, e.g., McCafferty etal., Nature
348:552-554 (1990);
Marks et al., Biotechnology 10:779-783 (1992)).
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[0134] The epitope of a mAb is the region of its antigen to which the mAb
binds. Two
antibodies bind to the same or overlapping epitope if each competitively
inhibits (blocks)
binding of the other to the antigen. That is, a lx, 5x, 10x, 20x or 100x
excess of one antibody
inhibits binding of the other by at least 30% but preferably 50%, 75%, 90% or
even 99% as
measured in a competitive binding assay (see, e.g., Junghans etal., Cancer
Res. 50:1495, 1990).
Alternatively, two antibodies have the same epitope if essentially all amino
acid mutations in the
antigen that reduce or eliminate binding of one antibody reduce or eliminate
binding of the other.
Two antibodies have overlapping epitopes if some amino acid mutations that
reduce or eliminate
binding of one antibody reduce or eliminate binding of the other.
[0135] For preparation of suitable antibodies of the invention and for use
according to the
invention, e.g., recombinant, monoclonal, or polyclonal antibodies, many
techniques known in
the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975);
Kozbor et al.,
Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies
and Cancer
Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology
(1991); Harlow &
Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal
Antibodies: Principles
and Practice (2d ed. 1986)). The genes encoding the heavy and light chains of
an antibody of
interest can be cloned from a cell, e.g., the genes encoding a monoclonal
antibody can be cloned
from a hybridoma and used to produce a recombinant monoclonal antibody. Gene
libraries
encoding heavy and light chains of monoclonal antibodies can also be made from
hybridoma or
plasma cells. Random combinations of the heavy and light chain gene products
generate a large
pool of antibodies with different antigenic specificity (see, e.g., Kuby,
Immunology (3rd ed.
1997)). Techniques for the production of single chain antibodies or
recombinant antibodies
(U.S. Patent 4,946,778, U.S. Patent No. 4,816,567) can be adapted to produce
antibodies to
polypeptides of this invention. Also, transgenic mice, or other organisms such
as other
mammals, may be used to express humanized or human antibodies (see, e.g., U.S.
Patent Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et
al., Bio/Technology
10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature
368:812-13
(1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger,
Nature
Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-
93 (1995)).
Alternatively, phage display technology can be used to identify antibodies and
heteromeric Fab
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fragments that specifically bind to selected antigens (see, e.g., McCafferty
et al., Nature 348:552-
554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can
also be made
bispecific, i.e., able to recognize two different antigens (see, e.g., WO
93/08829, Traunecker et
al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology
121:210 (1986)).
Antibodies can also be heteroconjugates, e.g., two covalently joined
antibodies, or immunotoxins
(see, e.g., U.S. Patent No. 4,676,980 , WO 91/00360; WO 92/200373; and EP
03089).
101361 Methods for humanizing or primatizing non-human antibodies are well
known in the art
(e.g., U.S. Patent Nos. 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761;
5,693,762;
5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87/02671; EP Patent
Application 0173494;
Jones et al. (1986) Nature 321:522; and Verhoyen et al. (1988) Science
239:1534). Humanized
antibodies are further described in, e.g., Winter and Milstein (1991) Nature
349:293. Generally,
a humanized antibody has one or more amino acid residues introduced into it
from a source
which is non-human. These non-human amino acid residues are often referred to
as import
residues, which are typically taken from an import variable domain.
Humanization can be
essentially performed following the method of Winter and co-workers (see,
e.g., Morrison et al.,
PNAS USA, 81:6851-6855 (1984), Jones et al,, Nature 321:522-525 (1986);
Riechmann et al.,
Nature 332:323-327 (1988); Morrison and 0i, Adv. Immunol., 44:65-92 (1988),
Verhoeyen et
al., Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-
596 (1992), Padlan,
Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217
(1994)), by
substituting rodent CDRs or CDR sequences for the corresponding sequences of a
human
antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S.
Patent No.
4,816,567), wherein substantially less than an intact human variable domain
has been substituted
by the corresponding sequence from a non-human species. In practice, humanized
antibodies are
typically human antibodies in which some CDR residues and possibly some FR
residues are
substituted by residues from analogous sites in rodent antibodies. For
example, polynucleotides
comprising a first sequence coding for humanized immunoglobulin framework
regions and a
second sequence set coding for the desired immunoglobulin complementarity
determining
regions can be produced synthetically or by combining appropriate cDNA and
genomic DNA
segments. Human constant region DNA sequences can be isolated in accordance
with well
known procedures from a variety of human cells.
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[0137] A "chimeric antibody" is an antibody molecule in which (a) the constant
region, or a
portion thereof, is altered, replaced or exchanged so that the antigen binding
site (variable
region) is linked to a constant region of a different or altered class,
effector function and/or
species, or an entirely different molecule which confers new properties to the
chimeric antibody,
e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion
thereof, is altered, replaced or exchanged with a variable region having a
different or altered
antigen specificity. The preferred antibodies of, and for use according to the
invention include
humanized and/or chimeric monoclonal antibodies.
[0138] Techniques for conjugating therapeutic agents to antibodies are well
known (see, e.g.,
Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer
Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56
(Alan R. Liss,
Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery"in Controlled Drug
Delivery (2nd
Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers
Of Cytotoxic Agents In Cancer Therapy: A Review" in Monoclonal Antibodies '84:
Biological
And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and
Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol.
Rev., 62:119-
58 (1982)). As used herein, the term "antibody-drug conjugate" or "ADC" refers
to a therapeutic
agent conjugated or otherwise covalently bound to to an antibody. A
"therapeutic agent" as
referred to herein, is a composition useful in treating or preventing a
disease such as cancer.
[0139] The phrase "specifically (or selectively) binds" to an antibody or
"specifically (or
selectively) immunoreactive with," when referring to a protein or peptide,
refers to a binding
reaction that is determinative of the presence of the protein, often in a
heterogeneous population
of proteins and other biologics. Thus, under designated immunoassay
conditions, the specified
antibodies bind to a particular protein at least two times the background and
more typically more
than 10 to 100 times background. Specific binding to an antibody under such
conditions requires
an antibody that is selected for its specificity for a particular protein. For
example, polyclonal
antibodies can be selected to obtain only a subset of antibodies that are
specifically
immunoreactive with the selected antigen and not with other proteins. This
selection may be
achieved by subtracting out antibodies that cross-react with other molecules.
A variety of
immunoassay formats may be used to select antibodies specifically
immunoreactive with a
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particular protein. For example, solid-phase ELISA immunoassays are routinely
used to select
antibodies specifically immunoreactive with a protein (see, e.g., Harlow &
Lane, Using
Antibodies, A Laboratory Manual (1998) for a description of immunoassay
formats and
conditions that can be used to determine specific immunoreactivity).
[0140] A "ligand" refers to an agent, e.g., a polypeptide or other molecule,
capable of binding
to a receptor.
[0141] "Contacting" is used in accordance with its plain ordinary meaning and
refers to the
process of allowing at least two distinct species (e.g. chemical compounds
including
biomolecules or cells) to become sufficiently proximal to react, interact or
physically touch. It
should be appreciated; however, the resulting reaction product can be produced
directly from a
reaction between the added reagents or from an intermediate from one or more
of the added
reagents which can be produced in the reaction mixture.
[0142] The term "contacting" may include allowing two species to react,
interact, or physically
touch, wherein the two species may be, for example, a pharmaceutical
composition as provided
herein and a cell or a pCREB detection agent as described herein and a pCREB
antigen. In
embodiments contacting includes, for example, allowing a pharmaceutical
composition as
described herein to interact with a cell or a patient. In further embodiments,
contacting includes,
for example, allowing a pCREB detection agent as described herein to interact
with a pCREB
antigen.
[0143] The terms "polypeptide, " "peptide" and "protein" are used
interchangeably herein to
refer to a polymer of amino acid residues, wherein the polymer may In
embodiments be
conjugated to a moiety that does not consist of amino acids. The terms apply
to amino acid
polymers in which one or more amino acid residue is an artificial chemical
mimetic of a
corresponding naturally occurring amino acid, as well as to naturally
occurring amino acid
polymers and non-naturally occurring amino acid polymers. A "fusion protein"
refers to a
chimeric protein encoding two or more separate protein sequences that are
recombinantly
expressed as a single moiety.
[0144] The term "peptidyl" and "peptidyl moiety" means a monovalent peptide.

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[0145] The term "amino acid" refers to naturally occurring and synthetic amino
acids, as
well as amino acid analogs and amino acid mimetics that function in a manner
similar to the
naturally occurring amino acids. Naturally occurring amino acids are those
encoded by the
genetic code, as well as those amino acids that are later modified, e.g.,
hydroxyproline, y-
carboxyglutamate, and 0-phosphoserine. Amino acid analogs refers to compounds
that have
the same basic chemical structure as a naturally occurring amino acid, i.e.,
an a carbon that is
bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g.,
homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs
have
modified R groups (e.g., norleucine) or modified peptide backbones, but retain
the same
basic chemical structure as a naturally occurring amino acid. Amino acid
mimetics refers to
chemical compounds that have a structure that is different from the general
chemical
structure of an amino acid, but that functions in a manner similar to a
naturally occurring
amino acid. The terms "non-naturally occurring amino acid" and "unnatural
amino acid"
refer to amino acid analogs, synthetic amino acids, and amino acid mimetics
which are not
found in nature.
[0146] Amino acids may be referred to herein by either their commonly known
three letter
symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical
Nomenclature Commission. Nucleotides, likewise, may be referred to by their
commonly
accepted single-letter codes.
[0147] "Conservatively modified variants" applies to both amino acid and
nucleic acid
sequences. With respect to particular nucleic acid sequences, "conservatively
modified variants"
refers to those nucleic acids that encode identical or essentially identical
amino acid sequences.
Because of the degeneracy of the genetic code, a number of nucleic acid
sequences will encode
any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode
the amino
acid alanine. Thus, at every position where an alanine is specified by a
codon, the codon can be
altered to any of the corresponding codons described without altering the
encoded polypeptide.
Such nucleic acid variations are "silent variations," which are one species of
conservatively
modified variations. Every nucleic acid sequence herein which encodes a
polypeptide also
describes every possible silent variation of the nucleic acid. One of skill
will recognize that each
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codon in a nucleic acid (except AUG, which is ordinarily the only codon for
methionine, and
TGG, which is ordinarily the only codon for tryptophan) can be modified to
yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic acid which
encodes a
polypeptide is implicit in each described sequence.
[0148] As to amino acid sequences, one of skill will recognize that individual
substitutions,
deletions or additions to a nucleic acid, peptide, polypeptide, or protein
sequence which alters,
adds or deletes a single amino acid or a small percentage of amino acids in
the encoded sequence
is a "conservatively modified variant" where the alteration results in the
substitution of an amino
acid with a chemically similar amino acid. Conservative substitution tables
providing
functionally similar amino acids are well known in the art. Such
conservatively modified
variants are in addition to and do not exclude polymorphic variants,
interspecies homologs, and
alleles of the invention.
[0149] The following eight groups each contain amino acids that are
conservative substitutions
for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins (1984)).
[0150] "Percentage of sequence identity" is determined by comparing two
optimally aligned
sequences over a comparison window, wherein the portion of the polynucleotide
or polypeptide
sequence in the comparison window may comprise additions or deletions (i.e.,
gaps) as
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compared to the reference sequence (which does not comprise additions or
deletions) for optimal
alignment of the two sequences. The percentage is calculated by determining
the number of
positions at which the identical nucleic acid base or amino acid residue
occurs in both sequences
to yield the number of matched positions, dividing the number of matched
positions by the total
number of positions in the window of comparison and multiplying the result by
100 to yield the
percentage of sequence identity.
[0151] The terms "identical" or percent "identity," in the context of two or
more nucleic acids
or polypeptide sequences, refer to two or more sequences or subsequences that
are the same or
have a specified percentage of amino acid residues or nucleotides that are the
same (i.e., 60%
identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity
over a
specified region, e.g., of the entire polypeptide sequences of the invention
or individual domains
of the polypeptides of the invention), when compared and aligned for maximum
correspondence
over a comparison window, or designated region as measured using one of the
following
sequence comparison algorithms or by manual alignment and visual inspection.
Such sequences
are then said to be "substantially identical." This definition also refers to
the complement of a
test sequence. Optionally, the identity exists over a region that is at least
about 50 nucleotides in
length, or more preferably over a region that is 100 to 500 or 1000 or more
nucleotides in length.
[0152] For sequence comparison, typically one sequence acts as a reference
sequence, to
which test sequences are compared. When using a sequence comparison algorithm,
test and
reference sequences are entered into a computer, subsequence coordinates are
designated, if
necessary, and sequence algorithm program parameters are designated. Default
program
parameters can be used, or alternative parameters can be designated. The
sequence comparison
algorithm then calculates the percent sequence identities for the test
sequences relative to the
reference sequence, based on the program parameters.
[0153] A "comparison window", as used herein, includes reference to a segment
of any one of
the number of contiguous positions selected from the group consisting of,
e.g., a full length
sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150
amino acids or
nucleotides in which a sequence may be compared to a reference sequence of the
same number
of contiguous positions after the two sequences are optimally aligned. Methods
of alignment of
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sequences for comparison are well-known in the art. Optimal alignment of
sequences for
comparison can be conducted, e.g., by the local homology algorithm of Smith
and Waterman
(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of
Needleman and
Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of
Pearson and Lipman
(1988) Proc. Nat'l. Acad Sci. USA 85:2444, by computerized implementations of
these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software

Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual
alignment
and visual inspection (see, e.g., Ausubel et al., Current Protocols in
Molecular Biology (1995
supplement)).
[0154] An example of an algorithm that is suitable for determining percent
sequence identity
and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are
described in
Altschul et at. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et at.
(1990)1 Mol. Biol.
215:403-410, respectively. Software for performing BLAST analyses is publicly
available
through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/).
This algorithm involves first identifying high scoring sequence pairs (HSPs)
by identifying short
words of length W in the query sequence, which either match or satisfy some
positive-valued
threshold score T when aligned with a word of the same length in a database
sequence. T is
referred to as the neighborhood word score threshold (Altschul et at., supra).
These initial
neighborhood word hits act as seeds for initiating searches to find longer
HSPs containing them.
The word hits are extended in both directions along each sequence for as far
as the cumulative
alignment score can be increased. Cumulative scores are calculated using, for
nucleotide
sequences, the parameters M (reward score for a pair of matching residues;
always > 0) and N
(penalty score for mismatching residues; always < 0). For amino acid
sequences, a scoring
matrix is used to calculate the cumulative score. Extension of the word hits
in each direction are
halted when: the cumulative alignment score falls off by the quantity X from
its maximum
achieved value; the cumulative score goes to zero or below, due to the
accumulation of one or
more negative-scoring residue alignments; or the end of either sequence is
reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and speed of the
alignment. The
BLASTN program (for nucleotide sequences) uses as defaults a word length (W)
of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences,
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the BLASTP program uses as defaults a word length of 3, and expectation (E) of
10, and the
BLOSLTM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad.
Sci. USA
89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a
comparison of both
strands.
[0155] The BLAST algorithm also performs a statistical analysis of the
similarity between two
sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA
90:5873-5787).
One measure of similarity provided by the BLAST algorithm is the smallest sum
probability
(P(N)), which provides an indication of the probability by which a match
between two nucleotide
or amino acid sequences would occur by chance. For example, a nucleic acid is
considered
similar to a reference sequence if the smallest sum probability in a
comparison of the test nucleic
acid to the reference nucleic acid is less than about 0.2, more preferably
less than about 0.01, and
most preferably less than about 0.001.
[0156] An indication that two nucleic acid sequences or polypeptides are
substantially
identical is that the polypeptide encoded by the first nucleic acid is
immunologically cross
reactive with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as
described below. Thus, a polypeptide is typically substantially identical to a
second polypeptide,
for example, where the two peptides differ only by conservative substitutions.
Another
indication that two nucleic acid sequences are substantially identical is that
the two molecules or
their complements hybridize to each other under stringent conditions, as
described below. Yet
another indication that two nucleic acid sequences are substantially identical
is that the same
primers can be used to amplify the sequence.
[0157] The term "isolated," when applied to a protein, denotes that the
protein is essentially
free of other cellular components with which it is associated in the natural
state. It is preferably
in a homogeneous state although it can be in either a dry or aqueous solution.
Purity and
homogeneity are typically determined using analytical chemistry techniques
such as
polyacrylamide gel electrophoresis or high performance liquid chromatography.
A protein that
is the predominant species present in a preparation is substantially purified.
The term "purified"
denotes that a protein gives rise to essentially one band in an
electrophoretic gel. Particularly, it

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means that the protein is at least 85% pure, more preferably at least 95%
pure, and most
preferably at least 99% pure.
[0158] The phrase "specifically (or selectively) binds" to an antibody or
"specifically (or
selectively) immunoreactive with," when referring to a protein or peptide,
refers to a binding
reaction that is determinative of the presence of the protein in a
heterogeneous population of
proteins and other biologics. Thus, under designated immunoassay conditions,
the specified
antibodies bind to a particular protein at least two times the background and
do not substantially
bind in a significant amount to other proteins present in the sample.
Typically a specific or
selective reaction will be at least twice background signal or noise and more
typically more than
to 100 times background.
[0159] A "cell" as used herein, refers to a cell carrying out metabolic or
other function
sufficient to preserve or replicate its genomic DNA. A cell can be identified
by well-known
methods in the art including, for example, presence of an intact membrane,
staining by a
particular dye, ability to produce progeny or, in the case of a gamete,
ability to combine with a
second gamete to produce a viable offspring. Cells may include prokaryotic and
eukaryotic
cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic
cells include but are
not limited to yeast cells and cells derived from plants and animals, for
example mammalian,
insect (e.g., spodoptera) and human cells.
[0160] As defined herein, the term "inhibition", "inhibit", "inhibiting" and
the like in reference
to a protein-inhibitor (e.g., an A2A receptor antagonist or a PD-1 signaling
pathway inhibitor)
interaction means negatively affecting (e.g., decreasing) the activity or
function of the protein
(e.g., decreasing the activity of an A2A receptor or a PD-1 protein or PD-Li
protein) relative to
the activity or function of the protein in the absence of the inhibitor (e.g.,
an A2A receptor
antagonist or a PD-1 signaling pathway inhibitor). In some embodiments,
inhibition refers to
reduction of a disease or symptoms of disease (e.g., cancer). Thus, inhibition
includes, at least in
part, partially or totally blocking stimulation, decreasing, preventing, or
delaying activation, or
inactivating, desensitizing, or down-regulating signal transduction or
enzymatic activity or the
amount of a protein (e.g., an A2A receptor or a PD-1 protein or PD-Li
protein). Similarly an
"inhibitor" is a compound or protein that inhibits an A2A receptor or a PD-1
protein or PD-Li
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protein, e.g.õ by binding, partially or totally blocking, decreasing,
preventing, delaying,
inactivating, desensitizing, or down-regulating activity (e.g., an A2A
receptor activity or a PD-1
protein activity or PD-Li protein activity).
[0161] An "anti-cancer agent" is a therapeutic used in the treatment or
prevention of cancer.
An anti-cancer agent can be a large or small molecule. Example anti-cancer
agents include
antibodies, small molecules, and large molecules or combinations thereof
[0162] "Anti-cancer agent" is used in accordance with its plain ordinary
meaning and refers to
a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having
antineoplastic
properties or the ability to inhibit the growth or proliferation of cells. In
some embodiments, an
anti-cancer agent is a chemotherapeutic. In some embodiments, an anti-cancer
agent is an agent
identified herein having utility in methods of treating cancer. In some
embodiments, an anti-
cancer agent is an agent approved by the FDA or similar regulatory agency of a
country other
than the USA, for treating cancer. Examples of anti-cancer agents include, but
are not limited to,
MEK (e.g. MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g. XL518, CI-1040,
PD035901,
selumetinib/ AZD6244, GSK1120212/ trametinib, GDC-0973, ARRY-162, ARRY-300,
AZD8330, PD0325901, U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766),
alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan,
melphalan,
mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards (e.g.,
mechloroethamine,
cyclophosphamide, chlorambucil, meiphalan), ethyl enimine and methylmelamines
(e.g.,
hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas
(e.g., carmustine,
lomusitne, semustine, streptozocin), triazenes (decarbazine)), anti-
metabolites (e.g., 5-
azathioprine, leucovorin, capecitabine, fludarabine, gemcitabine, pemetrexed,
raltitrexed, folic
acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil,
floxouridine,
Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin),
etc.), plant alkaloids
(e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin,
paclitaxel, docetaxel,
etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine,
etoposide (VP16),
etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g.,
doxorubicin, adriamycin,
daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone,
plicamycin, etc.),
platinum-based compounds or platinum containing agents (e.g. cisplatin,
oxaloplatin,
carboplatin), anthracenedione (e.g., mitoxantrone), substituted urea (e.g.,
hydroxyurea), methyl
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hydrazine derivative (e.g., procarbazine), adrenocortical suppressant (e.g.,
mitotane,
aminoglutethimide), epipodophyllotoxins (e.g., etoposide), antibiotics (e.g.,
daunorubicin,
doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), inhibitors of mitogen-
activated protein
kinase signaling (e.g. U0126, PD98059, PD184352, PD0325901, ARRY-142886,
SB239063,
SP600125, BAY 43-9006, wortmannin, or LY294002, Syk inhibitors, mTOR
inhibitors,
antibodies (e.g., rituxan), gossyphol, genasense, polyphenol E, Chlorofusin,
all trans-retinoic
acid (ATRA), bryostatin, tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL), 5-
aza-2'-deoxycytidine, all trans retinoic acid, doxorubicin, vincristine,
etoposide, gemcitabine,
imatinib (Gleevec®), geldanamycin, 17-N-Allylamino-17-
Demethoxygeldanamycin (17-
AAG), flavopiridol, LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412,
PD184352,
20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyluraci1; abiraterone; aclarubicin;
acylfulvene;
adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine;
ambamustine; amidox;
amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide;
anastrozole; andrographolide;
angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-
dorsalizing morphogenetic
protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;
antisense
oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis
regulators;
apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;
atrimustine;
axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine;
baccatin III
derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins;
benzoylstaurosporine;
beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF
inhibitor;
bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A;
bizelesin; breflate;
bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C;
camptothecin
derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole;
carboxyamidotriazole;
CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase
inhibitors (ICOS);
castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline
sulfonamide; cicaprost; cis-
porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B;
combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;
crisnatol;
cryptophycin 8; cryptophycin A derivatives; curacin A;
cyclopentanthraquinones; cycloplatam;
cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab;
decitabine;
dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane;
dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; 9-
dioxamycin;
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diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene;
dronabinol;
duocaintycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine;
elemene; emitefur;
epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen
antagonists;
etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;
fenretinide; filgrastim;
finasteride; flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride;
forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin;
gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione
inhibitors; hepsulfam;
heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin;
idoxifene;
idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;
immunostimulant peptides;
insulin-like growth factor-1 receptor inhibitor; interferon agonists;
interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;
isobengazole;
isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N
triacetate; lanreotide;
leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia
inhibiting factor;
leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin;
levami sole; liarozole;
linear polyamine analogue; lipophilic disaccharide peptide; lipophilic
platinum compounds;
lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine;
losoxantrone; lovastatin;
loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;
maitansine; mannostatin
A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix
metalloproteinase inhibitors;
menogaril; merbarone; meterelin; methioninase; metoclopramide; M1F inhibitor;
mifepristone;
miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;
mitolactol;
mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin;
mitoxantrone;
mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin;
monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug
resistance gene
inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer
agent; mycaperoxide B;
mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted
benzamides;
nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim;
nedaplatin;
nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin;
nitric oxide
modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide;
okicenone;
oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine
inducer;
ormaplatin; osaterone; oxaliplatin; oxaunomycin; palauamine;
palmitoylrhizoxin; pamidronic
acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;
peldesine; pentosan
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polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide;
perillyl alcohol;
phenazinomycin; phenyl acetate; phosphatase inhibitors; picibanil; pilocarpine
hydrochloride;
pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator
inhibitor; platinum
complex; platinum compounds; platinum-triamine complex; porfimer sodium;
porfiromycin;
prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors;
protein A-based
immune modulator; protein kinase C inhibitor; protein kinase C inhibitors,
microalgal; protein
tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors;
purpurins;
pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylerie conjugate; raf
antagonists;
raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras
inhibitors; ras-GAP
inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII
retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone Bl;
ruboxyl; safingol;
saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine;
senescence
derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors;
signal transduction
modulators; single chain antigen-binding protein; sizofuran; sobuzoxane;
sodium borocaptate;
sodium phenylacetate; solverol; somatomedin binding protein; sonermin;
sparfosic acid;
spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem
cell inhibitor; stem-
cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;
superactive vasoactive
intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic
glycosaminoglycans;
tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan
sodium; tegafur;
tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide;
tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline;
thrombopoietin; thrombopoietin
mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid
stimulating hormone;
tin ethyl etiopurpurin; tirapazamine; titanocene bi chloride; topsentin;
toremifene; totipotent stem
cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine;
trimetrexate; triptorelin;
tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC
inhibitors; ubenimex;
urogenital sinus-derived growth inhibitory factor; urokinase receptor
antagonists; vapreotide;
variolin B; vector system, erythrocyte gene therapy; velaresol; veramine;
verdins; verteporfin;
vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb;
zinostatin
stimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin,
acivicin; aclarubicin;
acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine;
ambomycin;
ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin;
asparaginase;

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asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;
bicalutamide; bisantrene
hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar
sodium;
bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer;
carboplatin;
carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil;
cirolemycin;
cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine;
daunorubicin
hydrochloride; decitabine; dexoimaplatin; dezaguanine; dezaguanine mesylate;
diaziquone;
doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate;
dromostanol one
propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin;
enloplatin;
enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin
hydrochloride;
estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide
phosphate;
etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine;
fludarabine phosphate;
fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine;
gemcitabine
hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; iimofosine;
interleukin Ii
(including recombinant interleukin II, or r1L₂), interferon alfa-2a;
interferon alfa-2b;
interferon alfa-nl; interferon alfa-n3; interferon beta-la; interferon gamma-
lb; iproplatin;
irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate;
liarozole
hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride;
masoprocol;
maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol
acetate;
melphalan; menogaril; mercaptopuiine; methotrexate; methotrexate sodium;
metoprine;
meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;
mitomycin;
mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie;
nogalamycin;
ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin
sulfate;
perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin;
plomestane;
porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride;
puromycin;
puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;
safingol hydrochloride;
semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium
hydrochloride;
spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur;
talisomycin; tecogalan sodium;
tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;
testolactone;
thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene
citrate; trestolone
acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate;
triptorelin; tubulozole
hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine
sulfate; vincristine
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sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate
sulfate; vinleurosine
sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzoli dine sulfate;
vorozole; zeniplatin;
zinostatin; zorubicin hydrochloride, agents that arrest cells in the G2-M
phases and/or modulate
the formation or stability of microtubules, (e.g. Taxol.TM (i.e. paclitaxel),
Taxotere.TM,
compounds comprising the taxane skeleton, Erbulozole (i.e. R-55104),
Dolastatin 10 (i.e. DLS-
and NSC-376128), Mivobulin isethionate (i.e. as CI-980), Vincristine, NSC-
639829,
Discodermolide (i.e. as NVP-XX-A-296), ABT-751 (Abbott, i.e. E-7010),
Altorhyrtins (e.g.
Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g. Spongistatin 1,
Spongistatin 2, Spongistatin
3, Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7,
Spongistatin 8, and Spongistatin
9), Cemadotin hydrochloride (i.e. LU-103793 and NSC-D-669356), Epothilones
(e.g. Epothilone
A, Epothilone B, Epothilone C (i.e. desoxyepothilone A or dEpoA), Epothilone D
(i.e. KOS-862,
dEpoB, and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone B N-
oxide, Epothilone
A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B (i.e. BMS-310705), 21-
hydroxyepothilone D (i.e. Desoxyepothilone F and dEpoF), 26-fluoroepothilone,
Auristatin PE
(i.e. NSC-654663), Soblidotin (i.e. TZT-1027)õ Vincristine sulfate,
Cryptophycin 52 (i.e. LY-
355703), Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (i.e. NSC-
106969), Oncocidin
Al (i.e. BTO-956 and DIIVIE), Fijianolide B, Laulimalide, Narcosine (also
known as NSC-5366),
Nascapine, Hemiasterlin, Vanadocene acetyl acetonate, Monsatrol, lnanocine
(i.e. NSC-698666),
Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin,
lsoeleutherobin A, and Z-
Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B, Diazonamide A,
Taccalonolide A,
Diozostatin, (-)-Phenylahistin (i.e. NSCL-96F037), Myoseverin B, Resverastatin
phosphate
sodium, steroids (e.g., dexamethasone), finasteride, aromatase inhibitors,
gonadotropin-releasing
hormone agonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids
(e.g.,
prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol
acetate,
medroxyprogesterone acetate), estrogens (e.g., di ethlystilbestrol, ethinyl
estradiol), anti estrogen
(e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone),
antiandrogen (e.g.,
flutamide), immunostimulants (e.g., Bacillus Calmette-Guerin (BCG), levami
sole, interleukin-2,
alpha-interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2,
anti-CD52, anti-
HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33
monoclonal
antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-pseudomonas
exotoxin
conjugate, etc.), radioimmunotherapy (e.g., anti-CD20 monoclonal antibody
conjugated to "In,
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90Y, or 1311, etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin,
epirubicin,
topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine,
sertraline,
pitavastatin, irinotecan, clofazimine, 5-nonyloxytryptamine, vemurafenib,
dabrafenib, erlotinib,
gefitinib, EGFR inhibitors, epidermal growth factor receptor (EGFR)-targeted
therapy or
therapeutic (e.g. gefitinib (Iressa TM), erlotinib (Tarceva TM), cetuximab
(ErbituxTm), lapatinib
(TykerbTm), panitumumab (VectibixTm), vandetanib (CaprelsaTm),
afatinib/BIBW2992, CI-
1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543,
ARRY-
380, AG-1478, dacomitinib/PF299804, OSI-420/desmethyl erlotinib, AZD8931,
AEE788,
pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035,
BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, hormonal therapies, or
the like.
[0163] "Analog" and "analogue" are used interchangeably and are used in
accordance with
their plain ordinary meaning within Chemistry and Biology and refers to a
chemical compound
that is structurally similar to another compound (i.e., a so-called
"reference" compound) but
differs in composition, e.g., in the replacement of one atom by an atom of a
different element, or
in the presence of a particular functional group, or the replacement of one
functional group by
another functional group, or the absolute stereochemistry of one or more
chiral centers of the
reference compound, including isomers thereof Accordingly, an analog is a
compound that is
similar or comparable in function and appearance but not in structure or
origin to a reference
compound. In embodiments, an analog is an adenosine analog.
[0164] An example adenosine analog is 5'-N-ethylcarboxamido-adenosine (NECA),
having the
structure shown below:
NH2
N
\)
H3C N, 0
OH OH
58

84342314
100091 As used herein, the term "about" means a range of values including the
specified value,
which a person of ordinary skill in the art would consider reasonably similar
to the specified
value. In embodiments, about means within a standard deviation using
measurements generally
acceptable in the art. In embodiments, about means a range extending to +1-
10% of the
specified value. In embodiments, about means the specified value.
100101 An "A2A receptor" or "adenosine A2A receptor" as referred to herein
includes any of
the recombinant or naturally-occurring forms of the adenosine A2A receptor
also known as
ADORA2A or variants or homologs thereof that maintain adenosine A2A receptor
activity (e.g.
within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity
compared to
adenosine A2A receptor). In some aspects, the variants or homologs have at
least 90%, 95%,
96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole
sequence or a
portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid
portion) compared to a
naturally occurring adenosine A2A receptor. In embodiments, the adenosine A2A
receptor is
substantially identical to the protein identified by the UniProt reference
number P29274 or a
variant or homolog having substantial identity thereto. In embodiments, the
adenosine A2A
receptor is substantially identical to the protein identified by the UniProt
reference number
Q60613 or a variant or homolog having substantial identity thereto.
100111 "A2B receptor" or "A2BR" or "Adenosine A2B receptor" are used
interchangeably.
A2B receptors are expressed in some mast cells, such as the BR line of canine
mastocytoma
cells, which appear to be responsible for triggering acute Ca' mobilization
and degranulation.
(See Auchampach et al., Mol. Pharmacol. 1997, 52, 846-S60 and Forsyth et al.,
Inflamm. Res.
1999, 48, 301-307.) Adenosine A2B receptors also trigger Ca" mobilization, and
participate in a
delayed IL8 release from human HMC-1 mast cells. Other functions associated
with the A2B AR
are the control of cell growth and gene expression, (See Neary et al., Trends
Neurosci. 1996, 19,
13-18.) endothelial-dependent vasodilation (See Martin et al., J Pharmacol.
Exp. Ther. 1993,
265, 248-2,53.), and fluid secretion from intestinal epithelia. (See
Strohmeier, et al., J Biol.
Chem. 1995, 270, 2387-2394.) Adenosine acting through A2B receptor subtype has
also been
reported to stimulate chloride permeability in cells expressing the cystic
fibrosis transport
regulator. (See Clancy et al., Am. J Physiol. 1999, 276, C361-C369.) Example
A2b receptor
antagonists are described in WO 2008002902. In
59
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embodiments, the adenosine A2b receptor is substantially identical to the
protein identified by
the UniProt reference number P29275 or a variant or homolog having substantial
identity thereto.
[0168] "Adenosine receptor agonist" refers to a molecule that activates
adenosine receptors
(e.g. A2a or A2b receptors). Adenosine receptors agonists can be small or
large molecule
agonists. Example adenosine receptors agonists include adenosine, NECA, or
analogs thereof.
[0169] "Adenosine receptor antagonist" references to a molecule that inhibits
activity of
adenosine receptors (e.g. A2a or A2b receptors). Adenosine receptors
antagonists can be small or
large molecule antagonists. In embodiments, CPI-444 is an example A2A receptor
antagonist.
[0170] A "PD-1 protein" or "PD-1" as referred to herein includes any of the
recombinant or
naturally-occurring forms of the Programmed cell death protein 1 (PD-1) also
known as cluster
of differentiation 279 (CD 279) or variants or homologs thereof that maintain
PD-1 protein
activity (e.g. within at least 500/0, 80%, 90%, 95%, 96%, 97%, 98%, 99% or
100% activity
compared to PD-1 protein). In some aspects, the variants or homologs have at
least 90%, 95%,
96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole
sequence or a
portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid
portion) compared to a
naturally occurring PD-1 protein. In embodiments, the PD-1 protein is
substantially identical to
the protein identified by the UniProt reference number Q15116 or a variant or
homolog having
substantial identity thereto. In embodiments, the PD-1 protein is
substantially identical to the
protein identified by the UniProt reference number Q02242 or a variant or
homolog having
substantial identity thereto.
[0171] A "pCREB protein" or "pCREB" as referred to herein includes any of the
recombinant
or naturally-occurring forms of the cAMP response element-binding protein
(pCREB) or variants
or homologs thereof that maintain pCREB activity (e.g. within at least 50%,
80%, 90%, 95%,
96%, 97%, 98%, 99% or 100% activity compared to pCREB protein). In some
aspects, the
variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino
acid
sequence identity across the whole sequence or a portion of the sequence (e.g.
a 50, 100, 150 or
200 continuous amino acid portion) compared to a naturally occurring pCREB
protein. In
embodiments, the pCREB protein is substantially identical to the protein
identified by the
UniProt reference number P16220 or a variant or homolog having substantial
identity thereto.

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[0172] A "PD-1 protein" or "PD-1" as referred to herein includes any of the
recombinant or
naturally-occurring forms of the Programmed cell death protein 1 (PD-1) also
known as cluster
of differentiation 279 (CD 279) or variants or homologs thereof that maintain
PD-1 protein
activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
activity
compared to PD-1 protein). In some aspects, the variants or homologs have at
least 90%, 95%,
96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole
sequence or a
portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid
portion) compared to a
naturally occurring PD-1 protein. In embodiments, the PD-1 protein is
substantially identical to
the protein identified by the UniProt reference number Q15116 or a variant or
homolog having
substantial identity thereto. In embodiments, the PD-1 protein is
substantially identical to the
protein identified by the UniProt reference number Q02242 or a variant or
homolog having
substantial identity thereto.
[0173] The term "atezolizumab" or "MPDL3280A" refers to a fully humanized,
engineered
monoclonal antibody of IgG1 isotype against the protein programmed cell death
ligand 1 (PD-
L1). In the customary sense, atezolizumab refers to CAS Registry number
1380723-44-3.
Atezolizumab may be referred to as an anti-cancer agent. In embodiments,
atezolizumab is
referred to as with the tradename Tecentriq .
[0174] Cyclic AMP (cAMP) response element binding protein (CREB) is a cellular

transcription factor. CREB is activated by signaling cascades resultant from
an array of
extracellular signals. One such activating signal cascade is triggered by
agonist binding to
adenosine receptor (e.g. A2A and A2B receptors). Agonist activation of
adenosine receptor
results in activation of CREB by phosphorylation. Agonist activation of
adenosine receptor also
results in activation of protein kinase A (PKA) upstream of CREB. In
embodiments, CREB is
substantially identical to the protein identified by the UniProt reference
number P16220 or a
variant or homolog having substantial identity thereto. In embodiments, CREB
is phosphorylated
at Serine 133.
[0175] A "pCREB detection agent" refers to a chemical or molecular moiety
capable of
identifying phosphorylated CREB. A pCREB detection agent may comprise an
antibody or
other specific for the phosphorylated CREB. Antibodies specific to pCREB are
commercially
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available, for example p-CREB Antibody (Ser133) (Cell Signaling Technology
Cat. No.: 14001
or Santa Cruz Biotechnology Cat. No.: sc-7978).
[0176] The term "blood cell detection agent" refers to a chemical or molecular
moiety capable
of identifying blood cells. A blood cell detection agent can refer to, for
example, a chemical
stain or an antibody against cell surface markers. Example blood cell
detection agents include B
cell detection agents and T cell detection agents.
[0177] The term "B cell detection agent" refers to a chemical or molecular
moiety capable of
identifying B cells. In examples, a B cell detection agent can be an antibody
to a B cell specific
surface maker (e.g. an antibody against CD19, or an antibody against CD20). B
cell detection
agents can be used alone or in combination. B cell detection agents can
further be detected by
fluorescence activated cell sorting (FACS).
[0178] "B Cells" or "B lymphocytes" refer to their standard use in the art. B
cells are
lymphocytes, a type of white blood cell (leukocyte), that develops into a
plasma cell (a "mature
B cell"), which produces antibodies. An "immature B cell" is a cell that can
develop into a
mature B cell. Generally, pro-B cells undergo immunoglobulin heavy chain
rearrangement to
become pro B pre B cells, and further undergo immunoglobulin light chain
rearrangement to
become an immature B cells. Immature B cells include Ti and T2 B cells.
[0179] "T cell detection agents" refers to a chemical or molecular moiety
capable of
identifying T cells. In examples, a T cell detection agent can be an antibody
to a T cell specific
surface maker (e.g. an antibody against CD3, and antibody against C4, or an
antibody against
CD8). T cell detection agents can be used alone or in combination. T cell
detection agents can
further be detected by fluorescence activated cell sorting (FACS).
[0180] "T cells" or "T lymphocytes" as used herein are a type of lymphocyte (a
subtype of
white blood cell) that plays a central role in cell-mediated immunity. They
can be distinguished
from other lymphocytes, such as B cells and natural killer cells, by the
presence of a T-cell
receptor on the cell surface. T cells include, for example, natural killer T
(NKT) cells, cytotoxic
T lymphocytes (CTLs), regulatory T (Treg) cells, and T helper cells. Different
types of T cells
can be distinguished by use of T cell detection agents.
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[0012] The term "cell subset detection agent" refers to a chemical or molecule
detection agent
that can be used to identify and distinguish a specific subset of cells (e.g.
senescent cells, naïve
cells, effector cells, memory cells etc). Example cell subset detection agents
include "naïve cell
detection agents", "memory cell detection agents", and "effector cell
detection agent." Cell
subset detection agents can include antibodies against distinguishing cell
surface markers. In
embodiments, cell subset detection agents include antibodies against CD27 or
antibodies against
CD45RA.
[0013] The term "apoptotic cells detection agent" refers to a chemical or
molecule detection
agent that can be used to identify and distinguish apoptotic cells. Apoptotic
cell detection agents
can include antibodies against distinguishing cell surface markers. An example
apoptosis
detection agent includes an antibody against cPARP. PARP is inactivated by
caspase cleavage.
Cleaved poly-ADP-ribose polymerase (PARP) (cPARP) is the cleavage product of
PARP.
cPARP can be used as a marker for apoptosis.
[0014] The term "CD" or "Cluster of Differentiation" refers to a nomenclature
system for
antigens found on lymphocytes, although CD antigens can be found on cells
other than
lymphocytes. This nomenclature is used to name antigens recognized by
monoclonal antibodies
that specifically bind an antigen on B cells, T cells or antigen presenting
cells. Each numeric
antigen is a specific protein that is recognized in the art by its CD
designation.
[0015] The term "CD3" as referred to herein is a protein complex comprising
four chains
including a CD3y chain, a CD3 5 chain, and two CD3E chains. An example
sequences of CD3
complex chains include: Epsilon chain precursor (GENBANK Accession No. NP
000724.1);
Gamma chain precursor (GENBANK6 Accession No. NP_000064.1); and Delta chain
precursor
(GENBANKO Accession No. NP 000723.1). Multiple isoforms are possible for each
of the
chains of CD3.
[0016] The terin "CD4" as referred to herein is a glycoprotein expressed on
the surface of T
helper cells, regulatory T cells, monocytes, macrophages, and dendritic cells.
CD4 was
originally known as leu-3 and T4 (after the OKT4 monoclonal antibody). CD4 as
referred to
herein has four immunoglobulin domains (Di to D4) that are exposed on the
extracellular surface
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of the cell, see ENTREZ No. 920, UNIPROT No. P01730, and GENBANKC Accession
No.
NP_000607.
[0017] A "CD4 + T lymphocyte" or "CD4 T cell" as referred to herein is
lymphocyte that
expresses the CD4 glycoprotein on its surface. CD4 T cells include helper T
cells, which are T
cells that help orchestrate the immune response, including antibody responses
and killer T cell
responses. CD4 T cell precursors differentiate into one of several subtypes,
including TH1 (type
1 helper T cell), TH2 (type 2 helper T cell), TH3 (T helper 3 cells), TH17 (T
helper 17 cells) or
'11-H (Follicular B helper T cells). These subtypes of helper T cells are
characterized by their
secretion of different cytokines to facilitate different types of immune
responses. In
embodiments, a CD4 T cell is an effector T cell. An "effector T cell" as
referred to herein is a T
cell that has been activated by its cognate antigen, and is actively involved
in eliminating a
pathogen. Thus, an effector T cell actively responds to a stimulus (a pathogen
or a co-
stimulation) and carries out a cell-mediated immune response. Non-limiting
examples of
effector T cells as referred to herein include helper T cells, killer T cells
(cytotoxic T cells) and
regulatory T cells.
[0018] The term "CD8" as referred to herein is a transmembrane glycoprotein
that serves as a
co-receptor for the T cell receptor (TCR). Like the TCR, CD8 binds to a major
histocompatibility complex (MHC) molecule, but is specific for the class I MHC
protein, see
ENTREZ No. 925 and UNIPROT No. P01732.
[0019] A "CD8 + T lymphocyte" or "CD8 T cell" as referred to herein is a
lymphocyte that
expresses the CD8 glycoprotein on its surface. Examples of CD8 T cells include
cytotoxic T
cells and natural killer cells. In one embodiment, a CD8T cell is a cytotoxic
T cell. In
embodiments, a CD8 T cell is a suppressor T cell.
[0020] CD20 is involved in regulating early steps in the activation and
differentiation process
of B cells (Tedder et al., Eur. J. Immunol. 16:881-887, 1986) and can function
as a calcium ion
channel (Tedder et al., J. Cell. Biochem.14D:195, 1990). Exemplary amino acid
sequences for
CD19 are provided in GENBANKC Accession Nos. NP_068769.2 (human), NP_690605.1
(human), and NP_031667.1 (mouse).
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[0021] CD27: A costimulatory immune checkpoint molecule. CD27 precursor
(human)(
GENBANK Accession No. NP 001233.1). Multiple isoforms exist.
[0022] The term "CD45RA" as provided herein refers to the CD45 Receptor
antigen also
known as Protein tyrosine phosphatase, receptor type, C (PTPRC). Exemplary
amino acid
sequences for CD45RA include GENBANK Accession Nos. NP 002829.3, NP 563578.2,

NP_ 563578.2, and NP 002829.3. CD45RA is expressed on naive T cells, as well
as on CD8-
and CD4-expressing effector cells. After antigen interaction, T cells gain
expression of
CD45R0 and lose expression of CD45RA. Thus, either CD45RA or CD45R0 is used to

generally differentiate the naive from memory T cell populations. Thus, a
"CD45RA-negative
CD8 T cell" as provided herein is a CD8 T cell which lacks expression of
detectable amounts of
CD45RA. In embodiments, the CD45RA-negative CD8 T cell is a memory T cell. A
"CD45RA-negative CD4 T cell" as provided herein is a CD4 T cell which lacks
expression of
detectable amounts of CD45RA. In embodiments, the CD45RA-negative CD4 T cell
is a
memory T cell. In embodiments, the CD45RA-negative CD8 T cell is a memory T
cell.
[0023] A "memory T cell" is a T cell that has previously encountered and
responded to its
cognate antigen during prior infection, encounter with cancer or previous
vaccination. At a
second encounter with its cognate antigen memory T cells can reproduce
(divide) to mount a
faster and stronger immune response than the first time the immune system
responded to the
pathogen. In embodiments, the memory T cell is a CD45RA-negative CD4 T cell.
In
embodiments, the memory T cell is a CD45RA-negative CD8 T cell.
[0024] A "regulatory T cell" or "suppressor T cell" is a lymphocyte which
modulates the
immune system, maintains tolerance to self-antigens, and prevents autoinunune
disease.
Regulatory T cells express the CD4, FOXP3, and CD25 and are thought to be
derived from the
same lineage as naive CD4 cells.
[0025] A "fixation agent" is a chemical or molecular agent capable of fixing a
cell (e.g. of
preserving a cell). A fixation agent can be used to prevent further biological
process in
preparation for cell staining, imaging or sorting. Fixation agents can be used
alone or in
combination. Non-limiting examples of fixation agents include formaldehyde,
glutaraldehyde,
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ethanol, methanol, Potassium dichromate, chromic acid, and potassium
permanganate, B-5,
Zenker's fixative, picrates, and HOPE.
[0195] A "cell permeabilizing agent" can include chemical or molecular agent,
or a
mechanical means of permeabilizing a cell. Non-limiting examples of
permeabilization agents
include organic solvents, such as methanol and acetone, and detergents such as
saponin, Triton
X-100 and Tween-20. The organic solvents dissolve lipids from cell membranes
making them
permeable to antibodies.
[0196] A "refractory subject" as provided herein is a subject that has been or
is being treated
for a disease or condition and does not respond to attempted forms of
treatment for said disease
or condition. For example, a cancer is said to be refractory when it does not
respond to (or is
resistant to) cancer treatment. A refractory cancer is also known as resistant
cancer. Thus, a
refractory subject is a subject that does not respond or is resistant to
treatment of a disease or
condition the subject is suffering from. In embodiments, a refractory subject
is a cancer patient
unresponsive to anti-PD-1 therapy. Where the cancer patient is unresponsive to
anti-PD-1
therapy the patient shows less than 20% reduction in tumor size or volume
after administration
of anti-PD-1 relative to a control. Thus, in embodiments, a refractory subject
shows less than
20% reduction in tumor size or volume after administration of anti-PD-1
relative to a control. In
embodiments, a refractory subject shows less than 10% reduction in tumor size
or volume after
administration of anti-PD-1 relative to a control. In embodiments, a
refractory subject shows
less than 5% reduction in tumor size or volume after administration of anti-PD-
1 relative to a
control. In embodiments, a refractory subject shows less than 1% reduction in
tumor size or
volume after administration of anti-PD-1 relative to a control. In
embodiments, a refractory
subject shows less than 0.5% reduction in tumor size or volume after
administration of anti-PD-1
relative to a control. In embodiments, a refractory subject shows less than
0.1% reduction in
tumor size or volume after administration of anti-PD-1 relative to a control.
[0197] The term "anti-tumor immune memory" as provided herein refers to the
ability of the
immune system of a subject to recognize (memorize) previously encountered
tumor antigen.
Once the tumor antigen has been recognized, the immune system reproduces
(e.g., through T cell
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activation and proliferation) and can mount a faster and stronger immune
response than the first
time it responded to the same tumor antigen.
[0198] The term "global immune activation" as provided herein refers to the
activation of
immune cells of the adaptive immune system in a subject. Examples of immune
cells activated
during global immune activation are without limitation, antigen presenting
cells (macrophages,
dendritic cells), B cells and T cells. The activation may occur through
recognition of a
previously encountered antigen (tumor antigen) or it may occur through
encounter of a novel
(not previously encountered) antigen (tumor antigen).
[0199] The terms "disease" or "condition" refer to a state of being or health
status of a patient
or subject capable of being treated with a compound, pharmaceutical
composition, or method
provided herein. In embodiments, the disease is cancer (e.g. lung cancer,
ovarian cancer,
osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer,
skin cancer (e.g.,
Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck
cancer, colorectal
cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer,
neuroblastoma). The
disease may be an autoimmune, inflammatory, cancer, infectious, metabolic,
developmental,
cardiovascular, liver, intestinal, endocrine, neurological, or other disease.
[0200] A "control" sample or value refers to a sample that serves as a
reference, usually a
known reference, for comparison to a test sample. For example, a test sample
can be taken from
a patient suspected of having a given disease (cancer) and compared to samples
from a known
cancer patient, or a known normal (non-disease) individual, A control can also
represent an
average value gathered from a population of similar individuals, e.g., cancer
patients or healthy
individuals with a similar medical background, same age, weight, etc. A
control value can also
be obtained from the same individual, e.g., from an earlier-obtained sample,
prior to disease, or
prior to treatment. One of skill will recognize that controls can be designed
for assessment of
any number of parameters.
[0201] One of skill in the art will understand which controls are valuable in
a given situation
and be able to analyze data based on comparisons to control values. Controls
are also valuable
for determining the significance of data. For example, if values for a given
parameter are widely
variant in controls, variation in test samples will not be considered as
significant.
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[0202] As used herein, the term "cancer" refers to all types of cancer,
neoplasm or malignant
tumors found in mammals, including leukemias, lymphomas, melanomas,
neuroendocrine
tumors, carcinomas and sarcomas. Exemplary cancers that may be treated with a
compound,
pharmaceutical composition, or method provided herein include lymphoma,
sarcoma, bladder
cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal
cancer, gastric
cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer,
leukemia, prostate
cancer, breast cancer (e.g. triple negative, ER positive, ER negative,
chemotherapy resistant,
herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen
resistant, ductal carcinoma,
lobular carcinoma, primary, metastatic), ovarian cancer, pancreatic cancer,
liver cancer
(e.g.hepatocellular carcinoma) , lung cancer (e.g. non-small cell lung
carcinoma, squamous cell
lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung
carcinoma,
carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate
cancer, castration-
resistant prostate cancer, breast cancer, triple negative breast cancer,
glioblastoma, ovarian
cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus),
colorectal cancer,
leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple
myeloma.
Additional examples include, cancer of the thyroid, endocrine system, brain,
breast, cervix,
colon, head & neck, esophagus, liver, kidney, lung, non-small cell lung,
melanoma,
mesothelioma, ovary, sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's
Disease, Non-
Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma
multiforme,
ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary
macroglobulinemia,
primary brain tumors, cancer, malignant pancreatic insulanoma, malignant
carcinoid, urinary
bladder cancer, premalignant skin lesions, testicular cancer, lymphomas,
thyroid cancer,
neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant
hypercalcemia,
endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or
exocrine pancreas,
medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal
cancer, papillary
thyroid cancer, hepatocellular carcinoma, Paget's Disease of the Nipple,
Phyllodes Tumors,
Lobular Carcinoma, Ductal Carcinoma, cancer of the pancreatic stellate cells,
cancer of the
hepatic stellate cells, or prostate cancer.
[0203] The term "leukemia" refers broadly to progressive, malignant diseases
of the blood-
forming organs and is generally characterized by a distorted proliferation and
development of
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leukocytes and their precursors in the blood and bone marrow. Leukemia is
generally clinically
classified on the basis of (1) the duration and character of the disease-acute
or chronic; (2) the
type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or
monocytic; and (3)
the increase or non-increase in the number abnormal cells in the blood-
leukemic or aleukemic
(subleukemic). Exemplary leukemias that may be treated with a compound,
pharmaceutical
composition, or method provided herein include, for example, acute
nonlymphocytic leukemia,
chronic lymphocytic leukemia, acute granulocytic leukemia, chronic
granulocytic leukemia,
acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia,
aleukocythemic
leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic
myelocytic
leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross'
leukemia, hairy-
cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic
leukemia, stem cell
leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia,
lymphoblastic
leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia,
lymphosarcoma
cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic
leukemia,
monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid
granulocytic
leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia,
multiple
myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia,
Schilling's
leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell
leukemia.
102041 The term "sarcoma" generally refers to a tumor which is made up of a
substance like
the embryonic connective tissue and is generally composed of closely packed
cells embedded in
a fibrillar or homogeneous substance. Sarcomas that may be treated with a
compound,
pharmaceutical composition, or method provided herein include a
chondrosarcoma,
fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma,
Abemethy's
sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma,
ameloblastic sarcoma,
botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma,
Wilms' tumor
sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial
sarcoma, fibroblastic
sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,
idiopathic multiple
pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma,
immunoblastic
sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma,
angiosarcoma,
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leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic
sarcoma,
Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectaltic
sarcoma.
[0205] The term "melanoma" is taken to mean a tumor arising from the
melanocytic system of
the skin and other organs. Melanomas that may be treated with a compound,
pharmaceutical
composition, or method provided herein include, for example, acral-lentiginous
melanoma,
amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91
melanoma,
Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma,
malignant
melanoma, nodular melanoma, subungal melanoma, or superficial spreading
melanoma.
[0206] The term "carcinoma" refers to a malignant new growth made up of
epithelial cells
tending to infiltrate the surrounding tissues and give rise to metastases.
Exemplary carcinomas
that may be treated with a compound, pharmaceutical composition, or method
provided herein
include, for example, medullary thyroid carcinoma, familial medullary thyroid
carcinoma, acinar
carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma,
carcinoma
adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell
carcinoma, basal
cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell
carcinoma,
bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma,
cerebriform
carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid
carcinoma, comedo
carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse,
carcinoma
cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,
ductal carcinoma,
carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid
carcinoma,
carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere,
carcinoma
fibrosum, gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma,
carcinoma
gigantocellul are, glandular carcinoma, granulosa cell carcinoma, hair-matrix
carcinoma,
hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline
carcinoma,
hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ,
intraepidermal
carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell
carcinoma,
large-cell carcinoma, lenticular carcinoma, carcinomalenticulare, lipomatous
carcinoma, lobular
carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary
carcinoma, melanotic
carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma

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myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma
ossificans, osteoid
carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma,
prickle cell
carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell
carcinoma,
carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma
scroti, signet-
ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid
carcinoma, spheroidal
cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous
carcinoma, squamous
cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma
telangiectodes,
transitional cell carcinoma, carcinoma tuberosum, tubular carcinoma, tuberous
carcinoma,
verrucous carcinoma, or carcinoma villosum.
[0207] As used herein, the terms "metastasis," "metastatic," and "metastatic
cancer" can be
used interchangeably and refer to the spread of a proliferative disease or
disorder, e.g., cancer,
from one organ or another non-adjacent organ or body part. Cancer occurs at an
originating site,
e.g., breast, which site is referred to as a primary tumor, e.g., primary
breast cancer. Some
cancer cells in the primary tumor or originating site acquire the ability to
penetrate and infiltrate
surrounding normal tissue in the local area and/or the ability to penetrate
the walls of the
lymphatic system or vascular system circulating through the system to other
sites and tissues in
the body. A second clinically detectable tumor formed from cancer cells of a
primary tumor is
referred to as a metastatic or secondary tumor. When cancer cells metastasize,
the metastatic
tumor and its cells are presumed to be similar to those of the original tumor.
Thus, if lung cancer
metastasizes to the breast, the secondary tumor at the site of the breast
consists of abnormal lung
cells and not abnormal breast cells. The secondary tumor in the breast is
referred to a metastatic
lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a
subject has or had
a primary tumor and has one or more secondary tumors. The phrases non-
metastatic cancer or
subjects with cancer that is not metastatic refers to diseases in which
subjects have a primary
tumor but not one or more secondary tumors. For example, metastatic lung
cancer refers to a
disease in a subject with or with a history of a primary lung tumor and with
one or more
secondary tumors at a second location or multiple locations, e.g., in the
breast.
102081 The term "associated" or "associated with" in the context of a
substance or substance
activity or function associated with a disease (e.g., diabetes, cancer (e.g.
prostate cancer, renal
cancer, metastatic cancer, melanoma, castration-resistant prostate cancer,
breast cancer, triple
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negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous
cell carcinoma (e.g.,
head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid
leukemia, lymphoma, B
cell lymphoma, or multiple myeloma)) means that the disease (e.g. lung cancer,
ovarian cancer,
osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer,
skin cancer (e.g.,
Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck
cancer, colorectal
cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer,
neuroblastoma) is caused by
(in whole or in part), or a symptom of the disease is caused by (in whole or
in part) the substance
or substance activity or function.
[0209] "Patient" or "subject in need thereof' refers to a living organism
suffering from or
prone to a disease or condition that can be treated by administration of a
composition or
pharmaceutical composition as provided herein. Non-limiting examples include
humans, other
mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and
other
non-mammalian animals. In some embodiments, a patient is human.
[0210] A patient or subject for the purposes of the present invention includes
both humans and
other animals, particularly mammals. Thus, the methods are applicable to both
human therapy
and veterinary applications. In the preferred embodiment the patient is a
mammal, preferably a
primate, and in the most preferred embodiment the patient is human.
METHODS OF TREATING CANCER
[0211] The methods provided herein are, inter alia, useful for the treatment
of cancer. Cancer
treatment can include administration of an anti-cancer agent. In embodiments,
an anti-cancer
agent includes an adenosine receptor antagonist, alone or or in combination.
In embodiments, an
adenosine receptor antagonist is an A2A receptor antagonist. Through
administration of a
therapeutically effective amount of an adenosine-A2A (A2A) receptor antagonist
alone or in
combination with a programmed cell death protein 1 (PD-1) signaling pathway
inhibitor cancer
may be treated in a subject in need thereof.
[0212] An "adenosine-A2A (A2A) receptor antagonist" as provided herein refers
to a
substance capable of detectably lowering expression or activity level of an
adenosine-A2A
(A2A) receptor compared to a control. The inhibited expression or activity of
the A2A receptor
can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less than that in a
control. In
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certain instances, the inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,
10-fold, or more in
comparison to a control. An "antagonist" is a compound or small molecule that
inhibits an A2A
receptor e.g., by binding, partially or totally blocking stimulation,
decrease, prevent, or delay
activation, or inactivate, desensitize, or down-regulate signal transduction,
gene expression or
enzymatic activity necessary for A2A activity. In embodiments, the A2A
receptor antagonist is a
compound or a small molecule. . In embodiments, the A2A receptor antagonist is
CPI-444. In
embodiments, the programmed cell death protein 1 (PD-1) signaling pathway
inhibitor is
atezolizumab. In embodiments, the A2A receptor antagonist and the PD-1
signaling pathway
inhibitor are administered simultaneously or sequentially.
[0213] Likewise, a "PD-1 signaling pathway inhibitor" as provided herein
refers to a substance
capable of detectably lowering expression of or activity level of the PD-1
signaling pathway
compared to a control. The inhibited expression or activity of the PD-1
signaling pathway can
be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less than that in a control.
In certain
instances, the inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-
fold, or more in comparison
to a control. An "inhibitor" is a compound or small molecule that inhibits the
PD-1 signaling
pathway e.g., by binding, partially or totally blocking stimulation of the PD-
1 signaling pathway,
decrease, prevent, or delay activation of the PD-1 signaling pathway, or
inactivate, desensitize,
or down-regulate signal transduction, gene expression or enzymatic activity of
the PD-1
signaling pathway. In embodiments, the PD-1 signaling pathway inhibitor
inhibits PD-1 activity
or expression. In embodiments, the PD-1 signaling pathway inhibitor inhibits
PD-Li activity or
expression. In embodiments, the PD-1 signaling pathway inhibitor is a compound
or a small
molecule. In embodiments, the PD-1 signaling pathway inhibitor is an antibody.
[0214] According to the methods provided herein, the subject is administered
an effective
amount of one or more of the agents (e.g., an A2A receptor antagonist and/or a
PD-1 signaling
pathway inhibitor) provided herein. An "effective amount" is an amount
sufficient to accomplish
a stated purpose (e.g. achieve the effect for which it is administered, treat
a disease (e.g., cancer),
reduce receptor signaling activity, reduce one or more symptoms of a disease
or condition). An
example of an "effective amount" is an amount sufficient to contribute to the
treatment,
prevention, or reduction of a symptom or symptoms of a disease (e.g., cancer),
which could also
be referred to as a "therapeutically effective amount." A "reduction" of a
symptom or symptoms
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(and grammatical equivalents of this phrase) means decreasing of the severity
or frequency of the
symptom(s), or elimination of the symptom(s). Guidance can be found in the
literature for
appropriate dosages for given classes of pharmaceutical products. For example,
for the given
parameter, a therapeutically effective amount will show an increase or
decrease of at least 5%,
10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. In
embodiments, this
increase or decrease for a given parameter may vary throughout the day (e.g. a
peak percentage
increase or decrease may differ from a percentage increase or decrease when
therapeutic
concentrations in circulating blood are at their peak or trough concentrations
dependent on daily
dosing patterns and individual pharmacokinetics). Efficacy can also be
expressed as "-fold"
increase or decrease. For example, a therapeutically effective amount can have
at least a 1.2-fold,
1.5-fold, 2-fold, 5-fold, or more effect over a control. The exact amounts
will depend on the
purpose of the treatment, and will be ascertainable by one skilled in the art
using known
techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3,
1992); Lloyd, The
Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar,
Dosage
Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th
Edition, 2003,
Gennaro, Ed., Lippincott, Williams & Wilkins).
[0215] Thus, in one aspect, a method of treating cancer in a subject in need
thereof is provided.
The method includes administering to the subject a therapeutically effective
amount of an
adenosine-A2A (A2A) receptor antagonist and a programmed cell death protein 1
(PD-1)
signaling pathway inhibitor.
[0216] In embodiments, the A2A receptor antagonist is a compound of formula:
R I
N,
N N R
(I).
[0217] In formula (I), R1 is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -SOr1R9,
_NHNH2, _0NR9Rio, _
NHC¨(0)NHNH2, ¨NHC¨(0)NR9Rio, _N(0).",_NR9Rto,
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-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9Rt ,o -0R9, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl.
[0218] R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -S0,12R11, -
S032NR11R12,
-NHNH2, -0NRIIR12, _NHC-(0)NHNH2, -NHC-(0)NR" "Rt2, _N(0).2,
_NRIIRt2, -C(0)R", -C(0)-OR", -C(0)NR1tR12, -OR", substituted ,
sunstitutea or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl.
[0219] R3 is independently hydrogen, halogen, -CX 3, -CN, -S02C1, -S0,13R13, -
S0,3NR13R14,
_NHNH2, _0N-R13Rt4, _NHc=(0)NHNH2, _NHc="NRt3R14, _N(0).13,
_Nee, _NH_O-R13, _c(0)-13, _
C(0)-0R13, -C(0)NR13R14, _0-13, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl.
[0220] R9, RI , RI", R12, Rt3 and R'4
are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -0001-1, -CR2COOH, -CONH2, -OR, -SH, -S02C1, -S03H, -SO4H, -
SO2NH2,
-NO2, -NHNH2, -ONH2, -NTIC=(0)NtINH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl. In
embodiments, R9, R10, R11, R12, R13 and K-14
are independently hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl.
[0221] Xa, Xb and Xc are independently -F, -Cl, -Br, or -I.
[0222] The symbols n1, n2 and n3 are independently an integer from 0 to 4. In
embodiments, ni
is 0. In embodiments, n1 is 1. In embodiments, n1 is 3. In embodiments, n1 is
4. In
embodiments, n2 is 0. In embodiments, n2 is 1. In embodiments, n2 is 3. In
embodiments, n2 is

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4. In embodiments, n3 is 0. In embodiments, n3 is 1. In embodiments, n3 is 3.
In embodiments,
n3 is 4.
[0223] The symbols mi, m2 and m3 are independently an integer from 1 to 2. In
embodiments,
mi is 0. In embodiments, m1 is 1. In embodiments, m1 is 2. In embodiments, m2
is 0. In
embodiments, m2 is 1. In embodiments, m2 is 2. In embodiments, m3 is 0. In
embodiments, m3
is 1. In embodiments, m2 is 2.
[0224] The symbols v1, v2 and v3 are independently an integer from 1 to 2. In
embodiments, v1
is 0. In embodiments, vi is 1. In embodiments, vi is 2. In embodiments, v2 is
0. In
embodiments, v2 is 1. In embodiments, v2 is 2. In embodiments, v3 is 0. In
embodiments, v3 is
1. In embodiments, v3 is 2.
[0225] In embodiments, RI is independently hydrogen, halogen, -CF3, -CN,
-CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2, -NO2,
-NH2, -NHNHz, -ONH2, -NHC¨(0)NHNH2, WA-substituted or unsubstituted alkyl, RIA-

substituted or unsubstituted heteroalkyl, R1A-substituted or unsubstituted
cycloalkyl, RIA-
substituted or unsubstituted heterocycloalkyl, RIA-substituted or
unsubstituted aryl, or RIA-
substituted or unsubstituted heteroaryl. RI may be RIA-substituted or
unsubstituted (e.g., CI-Cm
or Ci-C6) alkyl, RIA- substituted or unsubstituted (e.g., 2 to 20 membered or
2 to 6 membered)
heteroalkyl, RIA-substituted or unsubstituted (e.g., C3-C8 or C5-C7)
cycloalkyl, RIA-substituted or
unsubstituted (e.g., 3 to 8 membered or 3 to 6 membered) heterocycloalkyl, WA-
substituted or
unsubstituted (e.g., C5-C10 or C5-C6) aryl, or RIA-substituted or
unsubstituted (e.g., 5 to 10
membered or 5 to 6 membered) heteroaryl.
[0226] In embodiments, RA is independently hydrogen, halogen, =0, ¨S, -CF3, -
CN,
-CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2, -NO2,
-NH2, -NHNH2, -ONH2, -NIC¨(0)NHNH2, R1B-substituted or unsubstituted alkyl, Rm-

substituted or unsubstituted heteroalkyl, RIB-substituted or unsubstituted
cycloalkyl, RIB
-
substituted or unsubstituted heterocycloalkyl, RIB-substituted or
unsubstituted aryl, or RIB-
substituted or unsubstituted heteroaryl. RA may be RIB-substituted or
unsubstituted (e.g., CI-Czo
or CI-C6) alkyl, R113- substituted or unsubstituted (e.g., 2 to 20 membered or
2 to 6 membered)
heteroalkyl, RIB-substituted or unsubstituted (e.g., C3-C8 or C5-C7)
cycloalkyl, RIB-substituted or
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unsubstituted (e.g., 3 to 8 membered or 3 to 6 membered) heterocycloalkyl, WB-
substituted or
unsubstituted (e.g., C5-C10 or C5-C6) aryl, or R1B-substituted or
unsubstituted (e.g., 5 to 10
membered or 5 to 6 membered) heteroaryl.
[0227] In embodiments, RIB is independently hydrogen, halogen, =0, ¨S, -CF3, -
CN,
-CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2, -NO2,

-NH2, -NI-INH2, -ONH2, -NHC=(0)NHNH2, Ric-substituted or unsubstituted alkyl,
Ric-
substituted or unsubstituted heteroalkyl, Ric-substituted or unsubstituted
cycloalkyl, RC
substituted or unsubstituted heterocycloalkyl, Ric-substituted or
unsubstituted aryl, or Ric-
substituted or unsubstituted heteroaryl. R1B may be Ric-substituted or
unsubstituted (e.g., C1-C20
or CI-C6) alkyl, Ric- substituted or unsubstituted (e.g., 2 to 20 membered or
2 to 6 membered)
heteroalkyl, Ric-substituted or unsubstituted (e.g., C3-C8 or C5-C7)
cycloalkyl, Ric-substituted or
unsubstituted (e.g., 3 to 8 membered or 3 to 6 membered) heterocycloalkyl, Ric-
substituted or
unsubstituted (e.g., C5-C10 or C5-C6) aryl, or Ric-substituted or
unsubstituted (e.g., 5 to 10
membered or 5 to 6 membered) heteroaryl.
[0228] Ric is independently hydrogen, halogen, =0, ¨S, -CF3, -CN,
-CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2, -NO2,

-NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, unsubstituted alkyl, unsubstituted
heteroalkyl,
unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,
or unsubstituted
heteroaryl. Ric may be independently unsubstituted (e.g., C1-C20 or C1-C6)
alkyl, unsubstituted
(e.g., 2 to 20 membered or 2 to 6 membered) heteroalkyl, unsubstituted (e.g.,
C3-C8 or C5-C7)
cycloalkyl, unsubstituted (e.g., 3 to 8 membered or 3 to 6 membered)
heterocycloalkyl,
unsubstituted (e.g., C5-C10 or C5-C6) aryl, or unsubstituted (e.g., 5 to 10
membered or 5 to 6
membered) heteroaryl.
[0229] In embodiments, RI is independently R1A-substituted or unsubstituted
alkyl, WA-
substituted or unsubstituted heteroalkyl, WA-substituted or unsubstituted
cycloalkyl, RI A-
substituted or unsubstituted heterocycloalkyl, RIA-substituted or
unsubstituted aryl, or s R1A-
substituted or unsubstituted heteroaryl. In embodiments, le is WA-substituted
or unsubstituted
(e.g., 5 to 10 membered or 5 to 6 membered) heteroaryl. In embodiments, le is
unsubstituted 5
to 6 membered heteroaryl. In embodiments, R1 is RIA-substituted 5 to 6
membered heteroaryl.
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In embodiments, RI is unsubstituted 5 membered heteroaryl. In embodiments, RI
is WA-
substituted 5 membered heteroaryl. In embodiments, R1 is WA-substituted
furanyl.
[0230] In embodiments, R1A is R1B-substituted or unsubstituted (e.g., C1-C20
or C1-C6) alkyl.
In embodiments, R1A is R1B-substituted C1-C6 alkyl. In embodiments, RIA is
unsubstituted Ci-C6
alkyl. In embodiments, R1A is R1B-substituted C1-C4 alkyl. In embodiments, RA
is
unsubstituted CI-C.4 alkyl. In embodiments, R1A is R1B-substituted C1-C3
alkyl. In embodiments,
R1A is unsubstituted C1-C3 alkyl. In embodiments, R1A is methyl.
[0231] In embodiments, R2 is independently hydrogen,
halogen, -CXb3, -CN, -S02C1, -S0,12RI 1, -S0,2NR iRi2, 0NRiiR12,
-NHC=(0)NHNH2, -NHC=(0)NRiiR12, _N(0).2, _NRiiRi2,
-C(0)R11, -C(0)-OR", -C(0)NR"-K2

,

or -OR". In embodiments of the methods provided
herein, R2 is independently hydrogen, halogen, -CF3, -CN, -CC13, -COOH, -
CH2COOH,
-CONH2, -0H,-SH, -S02C1, -S03H, -SO4H, -SO2NH2, -NO2, -NH2, -NHNH2, -ONH2,
-NHC-(0)NHNH2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted
cycloalkyl,
unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted
heteroaryl. In embodiments,
R2 is -NRiiR12. In embodiments, R" and R.12 are independently hydrogen or
substituted or
unsubstituted (e.g., C1-C20 or CI-C6) alkyl. In embodiments, R" and R12 are
independently
substituted or unsubstituted C1-C6 alkyl. In embodiments, R" and R12 are
independently
substituted or unsubstituted C1-C4 alkyl. In embodiments, R" and R12 are
independently
substituted or unsubstituted C1-C3 alkyl. . In embodiments, R" and R12 are
independently
unsubstituted C1-C6 alkyl. In embodiments, R.11 and R12 are independently
substituted or
unsubstituted C1-C4 alkyl. In embodiments, R" and R12 are independently
unsubstituted C1-C3
alkyl. In embodiments, R" and R12 are independently hydrogen.
[0232] In embodiments, R3 is independently hydrogen, halogen, -CF3, -CN,
-CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2N1127 -
NO2,
- -NHNH2, -ONH2, -NHC=(0)NHNH2, R4-substituted or unsubstituted alkyl, R4-
substituted or unsubstituted heteroalkyl, WI-substituted or unsubstituted
cycloalkyl, R4-
substituted or unsubstituted heterocycloalkyl, WI-substituted or unsubstituted
aryl, or R4-
substituted or unsubstituted heteroaryl. R3 may be WI-substituted or
unsubstituted (e.g., C1-C20
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or C1-C6) alkyl, R4- substituted or unsubstituted (e.g., 2 to 20 membered or 2
to 6 membered)
heteroalkyl, R4-substituted or unsubstituted (e.g., C3-C8 or C5-C7)
cycloalkyl, R4-substituted or
unsubstituted (e.g., 3 to 8 membered or 3 to 6 membered) heterocycloalkyl, R4-
substituted or
unsubstituted (e.g., C5-C10 or C5-C6) aryl, or R4-substituted or unsubstituted
(e.g., 5 to 10
membered or 5 to 6 membered) heteroaryl.
[0233] R4 is independently hydrogen, halogen, =0, =S, -CF3, -CN,
-CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2, -NO2,

-NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, R5-substituted or unsubstituted alkyl, R5-
substituted or unsubstituted heteroalkyl, R5-substituted or unsubstituted
cycloalkyl, R5-
substituted or unsubstituted heterocycloalkyl, R5-substituted or unsubstituted
aryl, or R5-
substituted or unsubstituted heteroaryl. R4 may be R5-substituted or
unsubstituted (e.g., CI-C20
or C1-C6) alkyl, R5- substituted or unsubstituted (e.g., 2 to 20 membered or 2
to 6 membered)
heteroalkyl, R5-substituted or unsubstituted(e.g., C3-C8 or C5-C7) cycloalkyl,
R5-substituted or
unsubstituted (e.g., 3 to 8 membered or 3 to 6 membered) heterocycloalkyl, R5-
substituted or
unsubstituted (e.g., C5-C10 or C5-C6) aryl, or R5-substituted or unsubstituted
(e.g., 5 to 10
membered or 5 to 6 membered) heteroaryl.
[0234] R5 is independently hydrogen, halogen, =0, =S, -CF3, -CN,
-CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -S041-1, -SO2NH2, -
NO2,
-N142, -NHNfiz, -ONH2, -NHC=(0)NHNI-12, R6-substituted or unsubstituted alkyl,
R6-
substituted or unsubstituted heteroalkyl, R6-substituted or unsubstituted
cycloalkyl, R6-
substituted or unsubstituted heterocycloalkyl, R6-substituted or unsubstituted
aryl, or R6-
substituted or unsubstituted heteroaryl. R5 may be R6-substituted or
unsubstituted (e.g., Cl-Czo
or CI-C6) alkyl, R6- substituted or unsubstituted (e.g., 2 to 20 membered or 2
to 6 membered)
heteroalkyl, R6-substituted or unsubstituted (e.g., C3-C8 or C5-C7)
cycloalkyl, R6-substituted or
unsubstituted (e.g., 3 to 8 membered or 3 to 6 membered) heterocycloalkyl, R6-
substituted or
unsubstituted (e.g., C5-C10 or C5-C6) aryl, or R6-substituted or unsubstituted
(e.g., 5 to 10
membered or 5 to 6 membered) heteroaryl.
[0235] R6 is independently hydrogen, halogen, =0, =S, -CF3, -CN,
-CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2, -NO2,
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-NTINH2, -ONH2, -NHC=(0)NHNH2, R7-substituted or unsubstituted alkyl, R7-
substituted or unsubstituted heteroalkyl, R7-substituted or unsubstituted
cycloalkyl, R7-
substituted or unsubstituted heterocycloalkyl, R7-substituted or unsubstituted
aryl, or R7-
substituted or unsubstituted heteroaryl. R6 may be R7-substituted or
unsubstituted (e.g., C1-C20
or CI-C6) alkyl, R7- substituted or unsubstituted (e.g., 2 to 20 membered or 2
to 6 membered)
heteroalkyl, R7-substituted or unsubstituted (e.g., C3-C8 or C5-C7)
cycloalkyl, R7-substituted or
unsubstituted (e.g., 3 to 8 membered or 3 to 6 membered) heterocycloalkyl, R7-
substituted or
unsubstituted (e.g., C5-C10 or C5-C6) aryl, or R7-substituted or unsubstituted
(e.g., 5 to 10
membered or 5 to 6 membered) heteroaryl.
[0236] In embodiments, R3 is independently hydrogen, halogen, R4-substituted
or
unsubstituted alkyl, R4- substituted or unsubstituted heteroalkyl, R4-
substituted or unsubstituted
cycloalkyl, le-substituted or unsubstituted heterocycloalkyl, R4-substituted
or unsubstituted aryl,
or R4-substituted or unsubstituted heteroaryl. In embodiments, R3 is
independently R4-
substituted or unsubstituted (e.g., C1-C20 or CI-C6) alkyl. In embodiments, R3
is independently
R4-substituted or unsubstituted C1-C6 alkyl. In embodiments, R3 is
independently R4-substituted
or unsubstituted C1-05 alkyl. In embodiments, R3 is independently le-
substituted or
unsubstituted C1-C4 alkyl. In embodiments, R3 is independently R4-substituted
or unsubstituted
C1-C3 alkyl. In embodiments, R3 is independently unsubstituted C1-C6 alkyl. In
embodiments,
R3 is independently unsubstituted C1-05 alkyl. In embodiments, R3 is
independently R4-
unsubstituted CI-C.4 alkyl. In embodiments, R3 is independently unsubstituted
C1-C3 alkyl. In
embodiments, R3 is independently R4-substituted C1-C6 alkyl. In embodiments,
R3 is
independently R4-substituted C1-05 alkyl. In embodiments, R3 is independently
le-substituted
CI-CI alkyl. In embodiments, R3 is independently le-substituted C1-C3 alkyl.
In embodiments,
R3 is le-substituted C1 alkyl.
[0237] In embodiments, R4 is R5-substituted or unsubstituted (e.g., CI-Cm or
CI-C6) alkyl, R5-
substituted or unsubstituted (e.g., 2 to 20 membered or 2 to 6 membered)
heteroalkyl, R5-
substituted or unsubstituted(e.g., C3-C g or C5-C7) cycloalkyl, R5-substituted
or unsubstituted
(e.g., 3 to 8 membered or 3 to 6 membered) heterocycloalkyl, R5-substituted or
unsubstituted
(e.g., C5-C10 or C5-C6) aryl, or R5-substituted or unsubstituted (e.g., 5 to
10 membered or 5 to 6
membered) heteroaryl. In embodiments, R4 is R5-substituted or unsubstituted 5
to 6 membered

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heteroaryl. In embodiments, R4 is R5-substituted or unsubstituted 6 membered
heteroaryl. In
embodiments, R4 is unsubstituted 6 membered heteroaryl. In embodiments, R4 is
R5-substituted
6 membered heteroaryl. In embodiments, R4 is R5-substituted pyridinyl.
[0238] In embodiments, R5 is R6-substituted or unsubstituted (e.g., C1-C20 or
CI-C6) alkyl, R6-
substituted or unsubstituted (e.g., 2 to 20 membered or 2 to 6 membered)
heteroalkyl, R6-
substituted or unsubstituted (e.g., C3-C8 or C5-C7) cycloalkyl, R6-substituted
or unsubstituted
(e.g., 3 to 8 membered or 3 to 6 membered) heterocycloalkyl, R6-substituted or
unsubstituted
(e.g., C5-C10 or C5-C6) aryl, or R6-substituted or unsubstituted (e.g., 5 to
10 membered or 5 to 6
membered) heteroaryl. In embodiments, R5 is R6-substituted or unsubstituted 2
to 6 membered
heteroalkyl. In embodiments, R5 is R6-substituted or unsubstituted 2 to 5
membered heteroalkyl.
In embodiments, R5 is R6-substituted or unsubstituted 2 to 4 membered
heteroalkyl. In
embodiments, R5 is R6-substituted or unsubstituted 2 to 3 membered
heteroalkyl. In
embodiments, R5 is R6-substituted or unsubstituted 2 membered heteroalkyl. In
embodiments,
R5 is unsubstituted 2 to 6 membered heteroalkyl. In embodiments, R5 is
unsubstituted 2 to 5
membered heteroalkyl. In embodiments, R5 is unsubstituted 2 to 4 membered
heteroalkyl. In
embodiments, R5 unsubstituted 2 to 3 membered heteroalkyl. In embodiments, R5
is
unsubstituted 2 membered heteroalkyl. In embodiments, R5 is R6-substituted 2
to 6 membered
heteroalkyl. In embodiments, R5 is R6-substituted 2 to 5 membered heteroalkyl.
In
embodiments, R5 is R6-substituted 2 to 4 membered heteroalkyl. In embodiments,
R5 is R6-
substituted 2 to 3 membered heteroalkyl. In embodiments, R5 is R6-substituted
2 membered
heteroalkyl.
[0239] In embodiments, R6 is R7-substituted or unsubstituted (e.g., C1-C20 or
Ci-C6) alkyl, R7-
substituted or unsubstituted (e.g., 2 to 20 membered or 2 to 6 membered)
heteroalkyl, R7-
substituted or unsubstituted (e.g., C3-C8 or C5-C7) cycloalkyl, R7-substituted
or unsubstituted
(e.g., 3 to 8 membered or 3 to 6 membered) heterocycloalkyl, R7-substituted or
unsubstituted
(e.g., C5-C10 or C5-C6) aryl, or R7-substituted or unsubstituted (e.g., 5 to
10 membered or 5 to 6
membered) heteroaryl. In embodiments, R6 is R7-substituted or unsubstituted 3
to 6 membered
heterocycloalkyl. In embodiments, R6 is R7-substituted or unsubstituted 5
membered
heterocycloalkyl. In embodiments, R6 is R7-substituted 5 membered
heterocycloalkyl. In
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embodiments, R6 is unsubstituted 5 membered heterocycloalkyl. In embodiments,
R6 is
unsubstituted tetrahydrofuranyl.
[0240] In embodiments of the methods provided herein, R9, Rio, Ri2, Ri3 and
Ri4 are
independently hydrogen, halogen, =0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH,
-CONH2, -OH, -SH, -S02C1, -S03H, -SO4.11, -SO2NH2, -NO2, -NH2, -NHNH2, -ONH2,
-NHC-(0)NHNH2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted
cycloalkyl,
unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted
heteroaryl.
[0241] In embodiments, R1 is R1A-substituted furanyl. In one further
embodiment, R1A is
methyl. In another further embodiment, R2 is _NRii- 12.
In another further embodiment, R" and
R12 are independently hydrogen. In yet another further embodiment, R3 is R4-
substituted C1
alkyl. In another further embodiment, R4 is R5-substituted pyridinyl. In yet
another further
embodiment, R5 is R6-substituted 2 membered heteroalkyl. In another further
embodiments, 116
is unsubstituted tetrahydrofuranyl.
[0242] In embodiments, the A2A receptor antagonist is a compound of formula:
1,21
/
\NlµTT,1112
N
R62
R61 (II).
In formula (II), R6, R6-1 and R6-2 are independently hydrogen, halogen, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl. In
embodiments, R6, R6.1 and R6-2 are independently hydrogen, substituted or
unsubstituted alkyl,
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substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. In embodiments, R6.1 and R6.2 are hydrogen and R6 is a substituted
or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. In embodiments, R6.1 and R6'2 are hydrogen and R6 is
substituted or
unsubstituted heterocycloalkyl. In embodiments, R6-1 and R6-2 are hydrogen and
R6 is
unsubstituted heterocycloalkyl. In embodiments, le is substituted (e.g. with
an unsubstituted CI-
05 alkyl) or unsubstituted heteroaryl. In embodiments, le is substituted (e.g.
with an
unsubstituted C1-05 alkyl) or unsubstituted furanyl. In embodiments, R1 is
methyl-substituted
furanyl.
[0243] In formula (II), R1 and R6 are as described above (e.g., R6 may be R7-
substituted or
unsubstituted 3 to 6 membered heterocycloalkyl and R1 may be WA-substituted 5
to 6 membered
heteroaryl). Thus, in embodiments, R6 is unsubstituted tetrahydrofuranyl and
R1 is R1A-
substituted furanyl.
[0244] In formula (II), R6=1 may be independently hydrogen, halogen, -CF3, -
CN,
-CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2, -NO2,

-NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, R7-1-substituted or unsubstituted alkyl,
R7-1-
substituted or unsubstituted heteroalkyl, R7.1-substituted or unsubstituted
cycloalkyl, R7.1-
substituted or unsubstituted heterocycloalkyl, R7-1-substituted or
unsubstituted aryl, or R7'-
substituted or unsubstituted heteroaryl. R6.1 may be R7.1-substituted or
unsubstituted (e.g., C1-C20
or Cl-C6) alkyl, R7-1-- substituted or unsubstituted (e.g., 2 to 20 membered
or 2 to 6 membered)
heteroalkyl, R7.1-substituted or unsubstituted (e.g., C3-C8 or C5-C7)
cycloalkyl, R7.1-substituted or
unsubstituted (e.g., 3 to 8 membered or 3 to 6 membered) heterocycloalkyl, R7-
1-substituted or
unsubstituted (e.g., C5-C10 or C5-C6) aryl, or R7.1-substituted or
unsubstituted (e.g., 5 to 10
membered or 5 to 6 membered) heteroaryl. In embodiments, R6.1 is R7.1-
substituted or
unsubstituted C1-C6 alkyl. In embodiments, R6-1 is R7-1-substituted or
unsubstituted C1-05 alkyl.
In embodiments, R6.1 is R7.1-substituted or unsubstituted CI-C.4 alkyl. In
embodiments, R6.1 is
R7-1-substituted or unsubstituted C1-C3 alkyl. In embodiments, R6I is R7.1-
substituted C1-C6
alkyl. In embodiments, R6.1 is R7"-substituted C1-05 alkyl. In embodiments,
R6.1 is R7-1-
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substituted C1-C4 alkyl. In embodiments, R6.1 is R7.1-substituted C1-C3 alkyl.
In embodiments,
R6.1 is unsubstituted CI-C 6 alkyl. In embodiments, R6.1 is unsubstituted C1-
05 alkyl. In
embodiments, R6.1 is unsubstituted CI-C4 alkyl. In embodiments, R6.1 is
unsubstituted CI-C 3
alkyl. In embodiments, R6-1 is unsubstituted methyl.
[0245] R6.2 is independently hydrogen, halogen, =0, -CF3, -CN,
-CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -
SO2NH2, -NO2,
-NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, R7.2-substituted or unsubstituted alkyl,
R7.2-
substituted or unsubstituted heteroalkyl, R7.2-substituted or unsubstituted
cycloalkyl, R7.2-
substituted or unsubstituted heterocycloalkyl, R7-2-substituted or
unsubstituted aryl, or R7-2-
substituted or unsubstituted heteroaryl. R6.2 may be R72-substituted or
unsubstituted (e.g., C1-C20
or Ci-C6) alkyl, R7-2- substituted or unsubstituted (e.g., 2 to 20 membered or
2 to 6 membered)
heteroalkyl, R7.2-substituted or unsubstituted (e.g., C3-C8 or C 5 -C7)
cycloalkyl, R7.2-substituted or
unsubstituted (e.g., 3 to 8 membered or 3 to 6 membered) heterocycloalkyl, R7-
2-substituted or
unsubstituted (e.g., C5-C10 or C5-C6) aryl, or R7.2-substituted or
unsubstituted (e.g., 5 to 10
membered or 5 to 6 membered) heteroaryl. In embodiments, R6.2 is R7.2-
substituted or
unsubstituted C1-C6 alkyl. In embodiments, R6.2 is R7.2-substituted or
unsubstituted C1-05 alkyl.
In embodiments, R6.2 is R7.2-substituted or unsubstituted CI-CI alkyl. In
embodiments, R6.2 is
R7.2-substituted or unsubstituted CI-C 3 alkyl. In embodiments, R6.2 is R7.2-
substituted CI-C6
alkyl. In embodiments, R6.2 is R7.2-substituted C1-05 alkyl. In embodiments,
R62 is R7.2-
substituted C1-C4 alkyl. In embodiments, R6-2 is R7.2-substituted C1-C3 alkyl.
In embodiments,
R6.2 is unsubstituted C1-C6 alkyl. In embodiments, R6.2 is unsubstituted C1-05
alkyl. In
embodiments, R6.2 is unsubstituted CI-C.4 alkyl. In embodiments, R6.2 is
unsubstituted C1-C3
alkyl. In embodiments, R6.2 is unsubstituted methyl.
[0246] R7, R7-1 and R7-2 are independently hydrogen, halogen, =0, =S, -CF3, -
CN,
-CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -
SO2NH2, -NO2,
-NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, unsubstituted alkyl, unsubstituted
heteroalkyl,
unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,
or unsubstituted
heteroaryl. R7, R7.1 and R7-2 may be independently unsubstituted (e.g., C1-C20
or CI-C6) alkyl,
unsubstituted (e.g., 2 to 20 membered or 2 to 6 membered) heteroalkyl,
unsubstituted (e.g., C3-C8
or C5-C7) cycloalkyl, unsubstituted (e.g., 3 to 8 membered or 3 to 6 membered)
heterocycloalkyl,
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unsubstituted (e.g., C5-C10 or C5-C6) aryl, or unsubstituted (e.g., 5 to 10
membered or 5 to 6
membered) heteroaryl.
[0247] In embodiments, the A2A receptor antagonist is a compound of formula:
N =
N/
\ N N H2
0
[0248] In embodiments, the A2A receptor antagonist is a compound of formula:

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N,
NH2
=
[0249] In embodiments, the A2A receptor antagonist is a compound of formula:
cvo
o
1,TH2
________ N
0
. All compounds provided herein may optinally be
provided as a phaiinaceutically acceptable salt.
[0250] In embodiments, the PD-1 signaling pathway inhibitor is a programmed
death-ligand 1
(PD-L1) antagonist or a PD-1 antagonist. A PD-Li antagonist as provided herein
is a substance
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that, at least in part, partially or totally blocks stimulation, decreases,
prevents, or delays
activation, or inactivates, desensitizes, or down-regulates signal
transduction of PD-Li.
Likewise, a PD-1 antagonist as provided herein is a substance that, at least
in part, partially or
totally blocks stimulation, decreases, prevents, or delays activation, or
inactivates, desensitizes,
or down-regulates signal transduction of PD-1. In embodiments, the programmed
death-ligand 1
(PD-L1) antagonist is an antibody or a small molecule. In embodiments, the PD-
Li antagonist is
an antibody. In embodiments, the antibody is atezolizumab. The term
"atezolizumab" refers to a
fully humanized, engineered monoclonal antibody of IgG1 isotype against the
protein
programmed cell death ligand 1 (PD-L1). Atezolizumab is also known as
"MPDL3280A." In
the customary sense, atezolizumab refers to CAS Registry number 1380723-44-3.
[0251] In embodiments, the PD-1 antagonist is an antibody or a small molecule.
[0252] In embodiments, an adenosine receptor antagonists is administration in
conjunction
with an additional anti-cancer agent. In embodiments, an adenosine receptor
antagonist is
administered in conjunction with an antibody anti-cancer agent. In
embodiments, an adenosine
receptor antagonist is administered with a PD-Li antagonist. In embodiments,
an A2A receptor
antagonist is administered in conjunction with an antibody against PD-Li. In
embodiments, CPI-
444 is administered in conjunction with azetolizumab.
[0253] In embodiments, the A2A receptor antagonist and the PD-1 signaling
pathway inhibitor
are administered in a combined synergistic amount. A "combined synergistic
amount" as used
herein refers to the sum of a first amount (e.g., an amount of an A2A receptor
antagonist) and a
second amount (e.g., an amount of a PD-1 signaling pathway inhibitor) that
results in a
synergistic effect (i.e. an effect greater than an additive effect).
Therefore, the terms "synergy",
"synergism", "synergistic", "combined synergistic amount", and "synergistic
therapeutic effect"
which are used herein interchangeably, refer to a measured effect of compounds
administered in
combination where the measured effect is greater than the sum of the
individual effects of each
of the compounds administered alone as a single agent.
[0254] In embodiments, a synergistic amount may be about 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8,
0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,
2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0,
3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,
4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,
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5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,
6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,
7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,
9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,
9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the
amount of the A2A
receptor antagonist when used separately from the PD-1 signaling pathway
inhibitor. In
embodiments, a synergistic amount may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0,
Li, 1.2,1.3, 1.4, L5, L6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, 3.0, 3.1, 3.2,
3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,
4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,
5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9,
7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,
7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,
9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,
9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the amount of
the PD-1 signaling
pathway inhibitor when used separately from the A2A receptor antagonist.
[0255] The synergistic effect may be an A2A receptor activity decreasing
effect and/or a PD-1
signaling pathway activity decreasing effect. In embodiments, synergy between
the A2A
receptor antagonist and the PD-1 signaling pathway inhibitor may result in
about 0.1, 0.2, 0.3,
0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5,
2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,
4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,
4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2,
6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9,
7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4,
8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,
9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100%
greater decrease (e.g., decrease of A2A receptor activity or decrease of PD-1
signaling pathway
activity) than the sum of the decrease of the A2A receptor antagonist or the
PD-1 signaling
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pathway when used individually and separately. In embodiments, synergy between
the A2A
receptor antagonist and the PD-1 signaling pathway inhibitor may result in
0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,
2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,
2.8, 2.9, 3.0, 3..1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2,
4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,
5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,
7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,
8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,
9,4, 9,5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, or 100% greater
inhibition of the A2A receptor and/or the PD-1 signaling pathway than the sum
of the inhibition
of the A2A receptor antagonist or the PD-1 signaling pathway inhibitor when
used individually
and separately.
[0256] The synergistic effect may be a cancer-treating effect such as an lung
cancer (i.e. a lung
cancer-treating synergistic effect), bladder cancer (i.e. a bladder cancer-
treating synergistic
effect), melanoma (i.e. a melanoma-treating synergistic effect), renal cell
carcinoma (i.e. a renal
cell carcinoma-treating synergistic effect), colon cancer (i.e. a colon cancer-
treating synergistic
effect), ovarian cancer (i.e. an ovarian cancer-treating synergistic effect),
gastric cancer (i.e. a
gastric cancer-treating synergistic effect), breast cancer (i.e. a breast
cancer-treating synergistic
effect), head and neck carcinoma (i.e. a head and neck carcinoma-treating
synergistic effect),
prostate cancer (i.e. a prostate cancer-treating synergistic effect) and a
hematologic malignancy
(i.e. a hematologic malignancy-treating synergistic effect).
[0257] The A2A receptor antagonist and the PD-1 signaling pathway inhibitor
may be
administered in combination either concomitantly (e.g., as a mixture),
separately but
simultaneously (e.g., via separate intravenous lines) or sequentially (e.g.,
one agent is
administered first followed by administration of the second agent). Thus, the
term combination
is used to refer to concomitant, simultaneous or sequential administration of
the A2A receptor
antagonist and the PD-1 signaling pathway inhibitor. In embodiments, where the
A2A receptor
antagonist and the PD-1 signaling pathway inhibitor are administered
sequentially, the A2A
receptor antagonist is administered at a first time point and the PD-1
signaling pathway inhibitor
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is administered at a second time point, wherein the first time point precedes
the second time
point. The course of treatment is best determined on an individual basis
depending on the
particular characteristics of the subject and the type of treatment selected.
The treatment, such as
those disclosed herein, can be administered to the subject on a daily, twice
daily, bi-weekly,
monthly or any applicable basis that is therapeutically effective. The
treatment can be
administered alone or in combination with any other treatment disclosed herein
or known in the
art. The additional treatment can be administered simultaneously with the
first treatment, at a
different time, or on an entirely different therapeutic schedule (e.g., the
first treatment can be
daily, while the additional treatment is weekly). Thus, in embodiments, the
A2A receptor
antagonist and the PD-1 signaling pathway inhibitor are administered
simultaneously or
sequentially.
[0258] In embodiments, the A2A receptor antagonist is administered at a first
time point and
the PD-1 signaling pathway inhibitor is administered at a second time point,
wherein the first
time point precedes the second time point. In embodiments, the second time
point is within less
than about 120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10,
11, 9, 8, 7, 6, 5, 4, 3, 2
or 1 days from the first time point. In embodiments, the second time point is
within less than
about 120 days from the first time point. In embodiments, the second time
point is within less
than about 90 days from the first time point. In embodiments, the second time
point is within
less than about 60 days from the first time point. In embodiments, the second
time point is
within less than about 50 days from the first time point. In embodiments, the
second time point
is within less than about 40 days from the first time point. In embodiments,
the second time
point is within less than about 30 days from the first time point. In
embodiments, the second
time point is within less than about 20 days from the first time point.
[0259] In embodiments, the second time point is within less than about 19 days
from the first
time point. In embodiments, the second time point is within less than about 18
days from the
first time point. In embodiments, the second time point is within less than
about 17 days from
the first time point. hi embodiments, the second time point is within less
than about 16 days
from the first time point. In embodiments, the second time point is within
less than about 15
days from the first time point. In embodiments, the second time point is
within less than about
14 days from the first time point. In embodiments, the second time point is
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about 13 days from the first time point. In embodiments, the second time point
is within less
than about 12 days from the first time point. In embodiments, the second time
point is within
less than about 11 days from the first time point. In embodiments, the second
time point is
within less than about 10 days from the first time point. In embodiments, the
second time point
is within less than about 9 days from the first time point. In embodiments,
the second time point
is within less than about 8 days from the first time point. In embodiments,
the second time point
is within less than about 7 days from the first time point. In embodiments,
the second time point
is within less than about 6 days from the first time point. In embodiments,
the second time point
is within less than about 5 days from the first time point. In embodiments,
the second time point
is within less than about 4 days from the first time point. In embodiments,
the second time point
is within less than about 3 days from the first time point. In embodiments,
the second time point
is within less than about 2 days from the first time point. In embodiments,
the second time point
is within less than about 1 day from the first time point.
[0260] In embodiments, the second time point is within about 8, 10 or 12 days
from the first
time point. In embodiments, the second time point is within about 8, days from
the first time
point, hi embodiments, the second time point is within about 10 days from the
first time point.
In embodiments, the second time point is within about 12 days from the first
time point. In
embodiments, the PD-1 signaling pathway inhibitor and the A2A receptor
antagonist are
simultaneously administered at the second time point. In embodiments, the PD-1
signaling
pathway inhibitor and the A2A receptor antagonist are concomitantly
administered at the second
time point. In embodiments, the PD-1 signaling pathway inhibitor is
administered at the second
time point and the A2A receptor antagonist is not administered at the second
time point.
[0261] In embodiments, the PD-1 signaling pathway inhibitor is administered at
a first time
point and the A2A receptor antagonist is administered at a second time point,
wherein the first
time point precedes the second time point, hi embodiments, the second time
point is within less
than about 120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10,
11, 9, 8, 7, 6, 5, 4, 3, 2
or 1 days from the first time point. In embodiments, the second time point is
within less than
about 120 days from the first time point. In embodiments, the second time
point is within less
than about 90 days from the first time point. In embodiments, the second time
point is within
less than about 60 days from the first time point. In embodiments, the second
time point is
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within less than about 50 days from the first time point. In embodiments, the
second time point
is within less than about 40 days from the first time point. In embodiments,
the second time
point is within less than about 30 days from the first time point. In
embodiments, the second
time point is within less than about 20 days from the first time point.
[0262] In embodiments, the second time point is within less than about 19 days
from the first
time point. In embodiments, the second time point is within less than about 18
days from the
first time point. In embodiments, the second time point is within less than
about 17 days from
the first time point. In embodiments, the second time point is within less
than about 16 days
from the first time point. In embodiments, the second time point is within
less than about 15
days from the first time point. In embodiments, the second time point is
within less than about
14 days from the first time point. In embodiments, the second time point is
within less than
about 13 days from the first time point. In embodiments, the second time point
is within less
than about 12 days from the first time point. In embodiments, the second time
point is within
less than about 11 days from the first time point. In embodiments, the second
time point is
within less than about 10 days from the first time point. In embodiments, the
second time point
is within less than about 9 days from the first time point. In embodiments,
the second time point
is within less than about 8 days from the first time point. In embodiments,
the second time point
is within less than about 7 days from the first time point. In embodiments,
the second time point
is within less than about 6 days from the first time point. In embodiments,
the second time point
is within less than about 5 days from the first time point. In embodiments,
the second time point
is within less than about 4 days from the first time point. In embodiments,
the second time point
is within less than about 3 days from the first time point. In embodiments,
the second time point
is within less than about 2 days from the first time point. In embodiments,
the second time point
is within less than about 1 day from the first time point.
[0263] In embodiments, the second time point is within about 8, 10 or 12 days
from the first
time point. In embodiments, the second time point is within about 8, days from
the first time
point. In embodiments, the second time point is within about 10 days from the
first time point.
In embodiments, the second time point is within about 12 days from the first
time point. In
embodiments, the PD-1 signaling pathway inhibitor and the A2A receptor
antagonist are
simultaneously administered at the second time point. In embodiments, the PD-1
signaling
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pathway inhibitor and the A2A receptor antagonist are concomitantly
administered at the second
time point. In embodiments, the A2A receptor antagonist is administered at the
second time
point and the PD-1 signaling pathway inhibitor is not administered at the
second time point.
[0264] According to the methods provided herein, the subject is administered
an effective
amount of one or more of the agents (e.g., an A2A receptor antagonist and/or a
PD-1
signaling pathway inhibitor) provided herein. An "effective amount" is an
amount sufficient to
accomplish a stated purpose (e.g. achieve the effect for which it is
administered, treat a disease
(e.g., cancer), reduce receptor signalling activity, reduce one or more
symptoms of a disease or
condition). An example of an "effective amount" is an amount sufficient to
contribute to the
treatment, prevention, or reduction of a symptom or symptoms of a disease
(e.g., cancer), which
could also be referred to as a "therapeutically effective amount." A
"reduction" of a symptom or
symptoms (and grammatical equivalents of this phrase) means decreasing of the
severity or
frequency of the symptom(s), or elimination of the symptom(s). Guidance can be
found in the
literature for appropriate dosages for given classes of pharmaceutical
products. For example, for
the given parameter, a therapeutically effective amount will show an increase
or decrease of at
least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
Efficacy
can also be expressed as "-fold" increase or decrease. For example, a
therapeutically effective
amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect
over a control. The
exact amounts will depend on the purpose of the treatment, and will be
ascertainable by one
skilled in the art using known techniques (see, e.g., Liebernian,
Pharmaceutical Dosage Forms
(vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical
Compounding
(1999); Pickar, Dosage Calculations (1999); and Remington: The Science and
Practice of
Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).
[0265] In embodiments, the A2A receptor antagonist is administered at an
amount of about 0.5
mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30
mg/kg, 40
mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 200 mg/kg
or 300
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 0.5
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 1
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 5
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 10
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mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 20
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 30
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 40
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 50
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 60
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 70
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 80
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 90
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 100
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 200
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 300
mg/kg. It is understood that where the amount is referred to as "mg/kg", the
amount is milligram
per kilogram body weight of the subject being administered with the A2A
receptor antagonist.
[0266] In embodiments, the A2A receptor antagonist is administered at an
amount of about 0.5
mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60
mg/kg, 70
mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 200 mg/kg or 300 mg/kg. In embodiments,
the A2A
receptor antagonist is administered at an amount of about 1 mg/kg. In
embodiments, the A2A
receptor antagonist is administered at an amount of about 1 mg/kg to 2 mg/kg.
In embodiments,
the A2A receptor antagonist is administered at an amount of about 1 mg/kg to 3
mg/kg. In
embodiments, the A2A receptor antagonist is administered at an amount of about
1 mg/kg to 4
mg/kg. In embodiments, the A2A receptor antagonist is administered at an
amount of about 1
mg/kg to 5 mg/kg.
[0267] In embodiments, the A2A receptor antagonist is administered at an
amount of about 10
mg BID, 20 mg BID, 30 mg MD, 40 mg BID, 50 mg BID, 60 mg BID, 70 mg BID, 80 mg
BID,
90 mg BID, 100 mg MD, 110 mg BID, 120 mg BID, 130 mg BID, 140 mg BID, 150 mg
BID,
160 mg BID, 170 mg BID, 180 mg BID, 190 mg BID, 200 mg BID, 210 mg BID, 220 mg
BID,
230 mg BID, 240 mg BID, 250 mg MD, 260 mg MD, 270 mg BID, 280 mg BID, 290 mg
BID,
or 300 mg BID. In embodiments, the A2A receptor antagonist is administered at
an amount of
about 10 mg BID. In embodiments, the A2A receptor antagonist is administered
at an amount of
about 20 mg BID. In embodiments, the A2A receptor antagonist is administered
at an amount of
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about 30 mg BID. In embodiments, the A2A receptor antagonist is administered
at an amount of
about 40 mg BID. In embodiments, the A2A receptor antagonist is administered
at an amount of
about 50 mg BID. In embodiments, the A2A receptor antagonist is administered
at an amount of
about 60 mg BID. In embodiments, the A2A receptor antagonist is administered
at an amount of
about 70 mg BID. In embodiments, the A2A receptor antagonist is administered
at an amount of
about 80 mg BID. In embodiments, the A2A receptor antagonist is administered
at an amount of
about 90 mg BID. In embodiments, the A2A receptor antagonist is administered
at an amount of
about 100 mg BID. It is understood that where the amount is referred to as
"BID" which stands
for "bis in die", the amount is administered twice a day.
[0268] In embodiments, the A2A receptor antagonist is administered at an
amount of about
110 mg BID. In embodiments, the A2A receptor antagonist is administered at an
amount of
about 120 mg BID. In embodiments, the A2A receptor antagonist is administered
at an amount
of about 130 mg BID. In embodiments, the A2A receptor antagonist is
administered at an
amount of about 140 mg BID. In embodiments, the A2A receptor antagonist is
administered at
an amount of about 150 mg BID. In embodiments, the A2A receptor antagonist is
administered
at an amount of about 160 mg BID. In embodiments, the A2A receptor antagonist
is
administered at an amount of about 170 mg BID. In embodiments, the A2A
receptor antagonist
is administered at an amount of about 180 mg BID. In embodiments, the A2A
receptor
antagonist is administered at an amount of about 190 mg BID. In embodiments,
the A2A
receptor antagonist is administered at an amount of about 200 mg BID. It is
understood that
where the amount is referred to as "BID" which stands for "bis in die", the
amount is
administered twice a day.
[0269] In embodiments, the A2A receptor antagonist is administered at an
amount of about
210 mg BID. In embodiments, the A2A receptor antagonist is administered at an
amount of
about 220 mg BID. In embodiments, the A2A receptor antagonist is administered
at an amount
of about 230 mg BID. In embodiments, the A2A receptor antagonist is
administered at an
amount of about 240 mg BID. In embodiments, the A2A receptor antagonist is
administered at
an amount of about 250 mg BID. In embodiments, the A2A receptor antagonist is
administered
at an amount of about 260 mg BID. In embodiments, the A2A receptor antagonist
is
administered at an amount of about 270 mg BID. In embodiments, the A2A
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is administered at an amount of about 280 mg BID. In embodiments, the A2A
receptor
antagonist is administered at an amount of about 290 mg BID. In embodiments,
the A2A
receptor antagonist is administered at an amount of about 300 mg BID. It is
understood that
where the amount is referred to as "BID" which stands for "bis in die", the
amount is
administered twice a day.
[0270] In embodiments, the A2A receptor antagonist is administered at an
amount of about 10
mg QD, 20 mg QD, 30 mg QD, 40 mg QD, 50 mg QD, 60 mg QD, 70 mg QD, 80 mg QD,
90
mg QD, 100 mg QD, 110 mg QD, 120 mg QD, 130 mg QD, 140 mg QD, 150 mg QD, 160
mg
QD, 170 mg QD, 180 mg QD, 190 mg QD, 200 mg QD, 210 mg QD, 220 mg QD, 230 mg
QD,
240 mg QD, 250 mg QD, 260 mg QD, 270 mg QD, 280 mg QD, 290 mg QD, or 300 mg
QD. In
embodiments, the A2A receptor antagonist is administered at an amount of about
10 mg Q.D. In
embodiments, the A2A receptor antagonist is administered at an amount of about
20 mg QD. In
embodiments, the A2A receptor antagonist is administered at an amount of about
30 mg QD. In
embodiments, the A2A receptor antagonist is administered at an amount of about
40 mg QD. In
embodiments, the A2A receptor antagonist is administered at an amount of about
50 mg QD. In
embodiments, the A2A receptor antagonist is administered at an amount of about
60 mg QD. In
embodiments, the A2A receptor antagonist is administered at an amount of about
70 mg QD. In
embodiments, the A2A receptor antagonist is administered at an amount of about
80 mg QD. In
embodiments, the A2A receptor antagonist is administered at an amount of about
90 mg QD. In
embodiments, the A2A receptor antagonist is administered at an amount of about
100 mg QD. It
is understood that where the amount is referred to as "QD" which stands for
"quaque die", the
amount is administered once a day.
[0271] In embodiments, the A2A receptor antagonist is administered at an
amount of about
110 mg QD. In embodiments, the A2A receptor antagonist is administered at an
amount of
about 120 mg QD. In embodiments, the A2A receptor antagonist is administered
at an amount
of about 130 mg QD. In embodiments, the A2A receptor antagonist is
administered at an
amount of about 140 mg QD. In embodiments, the A2A receptor antagonist is
administered at
an amount of about 150 mg QD. In embodiments, the A2A receptor antagonist is
administered
at an amount of about 160 mg QD. In embodiments, the A2A receptor antagonist
is
administered at an amount of about 170 mg QD. In embodiments, the A2A receptor
antagonist
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is administered at an amount of about 180 mg QD. In embodiments, the A2A
receptor
antagonist is administered at an amount of about 190 mg QD. In embodiments,
the A2A
receptor antagonist is administered at an amount of about 200 mg QD. It is
understood that
where the amount is referred to as "QD" which stands for "quaque die", the
amount is
administered once a day.
[0272] In embodiments, the A2A receptor antagonist is administered at an
amount of about
210 mg QD. In embodiments, the A2A receptor antagonist is administered at an
amount of
about 220 mg QD. In embodiments, the A2A receptor antagonist is administered
at an amount
of about 230 mg QD. In embodiments, the A2A receptor antagonist is
administered at an
amount of about 240 mg QD. In embodiments, the A2A receptor antagonist is
administered at
an amount of about 250 mg QD. In embodiments, the A2A receptor antagonist is
administered
at an amount of about 260 mg QD. In embodiments, the A2A receptor antagonist
is
administered at an amount of about 270 mg QD. In embodiments, the A2A receptor
antagonist
is administered at an amount of about 280 mg QD. In embodiments, the A2A
receptor
antagonist is administered at an amount of about 290 mg QD. In embodiments,
the A2A
receptor antagonist is administered at an amount of about 300 mg QD. It is
understood that
where the amount is referred to as "QD" which stands for "quaque die", the
amount is
administered once a day.
[0273] The A2A receptor antagonist may be administered at an amount as
provided herein on
28 consecutive days. The A2A receptor antagonist may be administered at an
amount as
provided herein on 14 consecutive days. In embodiments, the A2A receptor
antagonist is
administered at 50mg BID, 100mg BID or 200mg QD. In embodiments, the A2A
receptor
antagonist is administered at 50mg BID. In embodiments, the A2A receptor
antagonist is
administered at 100mg BID. In embodiments, the A2A receptor antagonist is
administered at
200mg QD. In embodiments, the A2A receptor antagonist is administered at 100mg
BID and the
PD-1 signaling pathway inhibitor is administered at an amount of 840 mg. In
further
embodiments, the A2A receptor antagonist and the PD-1 signaling pathway
inhibitor are
administered simultaneously on 28 consecutive days. In other further
embodiments, the A2A
receptor antagonist and the PD-1 signaling pathway inhibitor are administered
simultaneously on
14 consecutive days.
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[0274] In embodiments, the PD-1 signaling pathway inhibitor is administered at
an amount of
less than about 1,300 mg. In embodiments, the PD-1 signaling pathway inhibitor
is administered
at an amount of less than about 1,200 mg. In embodiments, the PD-1 signaling
pathway
inhibitor is administered at an amount of less than about 1,100 mg. In
embodiments, the PD-1
signaling pathway inhibitor is administered at an amount of less than about
1,000 mg. In
embodiments, the PD-1 signaling pathway inhibitor is administered at an amount
of less than
about 900 mg. In embodiments, the PD-1 signaling pathway inhibitor is
administered at an
amount of less than about 800 mg. In embodiments, the PD-1 signaling pathway
inhibitor is
administered at an amount of less than about 700 mg. In embodiments, the PD-1
signaling
pathway inhibitor is administered at an amount of less than about 600 mg. In
embodiments, the
PD-1 signaling pathway inhibitor is administered at an amount of less than
about 500 mg. In
embodiments, the PD-1 signaling pathway inhibitor is administered at an amount
of less than
about 400 mg. In embodiments, the PD-1 signaling pathway inhibitor is
administered at an
amount of less than about 300 mg. In embodiments, the PD-1 signaling pathway
inhibitor is
administered at an amount of less than about 200 mg. In embodiments, the PD-1
signaling
pathway inhibitor is administered at an amount of less than about 100 mg. In
embodiments, the
PD-1 signaling pathway inhibitor is administered at an amount of about 100 mg,
200 mg, 300
mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,00 mg, 1,100 mg, 1,200
mg, or 1,300
mg. It is understood that where the amount is referred to as "mg" that the
amount is the total
amount in milligram of PD-1 signaling pathway inhibitor administered to the
subject.
[0275] In embodiments, the PD-1 signaling pathway inhibitor is administered at
an amount of
about 700 mg. In embodiments, the PD-1 signaling pathway inhibitor is
administered at an
amount of about 720 mg. In embodiments, the PD-1 signaling pathway inhibitor
is administered
at an amount of about 740 mg. In embodiments, the PD-1 signaling pathway
inhibitor is
administered at an amount of about 760 mg. In embodiments, the PD-1 signaling
pathway
inhibitor is administered at an amount of about 780 mg. In embodiments, the PD-
1 signaling
pathway inhibitor is administered at an amount of about 800 mg. In
embodiments, the PD-1
signaling pathway inhibitor is administered at an amount of about 820 mg. In
embodiments, the
PD-1 signaling pathway inhibitor is administered at an amount of about 840 mg.
In
embodiments, the PD-1 signaling pathway inhibitor is administered at an amount
of about 860
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mg. In embodiments, the PD-1 signaling pathway inhibitor is administered at an
amount of
about 880 mg. In embodiments, the PD-1 signaling pathway inhibitor is
administered at an
amount of about 900 mg. It is understood that where the amount is referred to
as "mg" that the
amount is the total amount in milligram of PD-1 signaling pathway inhibitor
administered to the
subject.
[0276] The methods provided herein are, inter alia, useful for the treatment
of cancer. In
embodiments, the cancer is selected from lung cancer, bladder cancer,
melanoma, renal cell
carcinoma, colon cancer, ovarian cancer, gastric cancer, breast cancer, head
and neck carcinoma,
prostate cancer and a hematologic malignancy. In embodiments, the cancer is
lung cancer. In
embodiments, the cancer is bladder cancer. In embodiments, the cancer is
melanoma. In
embodiments, the cancer is renal cell carcinoma. In embodiments, the cancer is
colon cancer. In
embodiments, the cancer is ovarian cancer. In embodiments, the cancer is
gastric cancer. In
embodiments, the cancer is breast cancer. In embodiments, the cancer is head
and neck
carcinoma. In embodiments, the cancer is prostate cancer. In embodiments, the
cancer is a
hematologic malignancy.
[0277] In another aspect, a method of treating cancer in a subject in need
thereof is provided.
The method includes administering to the subject a therapeutically effective
amount of an
adenosine-A2A (A2A) receptor antagonist of formula:
RI
2
R3 (I).
[0278] In formula (I), R1 is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -SOr1R9,
-SOvINR9Rto, _NHNH2, _0NR9Rio, _NFIC=(0)NHNH2, ¨NTC=(0)NR9Rto,
_N(0).1,_NR9Rio,
-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9R1o, -0R9, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl.
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[0279] R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -S0n2R11, -
S0,2NR11R12,
_NHNH2, _coNRitRu, _NHc=(0)NHNH2, _NHc=(0)NittiRt2, _N(0).12,
_NR11- 12,
NH-O-R", -C(0)R11, -C(0)-0R11, -C(0)NRIIR12, _0-11
.tc;
substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl.
[0280] R3 is independently hydrogen, halogen, -CX 3, -CN, -S02C1, -S0,0R13, -
S0,3NR13R14,
-NHNH2, -0NRI3R14, -NHC=(0)NHNH2, -NHC=(0)NRI3R14, -N(0)õ,3,
_NR13R147 _c(o)R13, _
C(0)-0R13, -C(0)NRI3R14, , ,
substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl.
[0281] R9, Rlo, RI% R12, R13 and R'4
are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NHNH2, -ONH2, -NHC=(0)1\11-1NH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
[0282] Xa, Xb and Xc are independently -F, -Cl, -Br, or -I.
[0283] ni, n2 and n3 are independently an integer from 0 to 4.
[0284] mi, m2 and m3 are independently an integer from 1 to 2.
[0285] And vi, v2 and v3 are independently an integer from 1 to 2.
[0286] The A2A receptor antagonist provided herein is the same A2A receptor
antagonist as
described above for aspects of treating cancer using an A2A receptor
antagonist and a PD-1
signaling pathway inhibitor. Therefore, the definitions for substituents and
variables of formula
(I) and (II) are the same as described above (e.g., RI is RIA-substituted
furanyl; RA is methyl; R2
is _NRIIR12; Rn and K-12
are independently hydrogen; R3 is R4-substituted C1 alkyl; R4 is R5-
substituted pyridinyl; R5 is R6-substituted 2 membered heteroalkyl; R6 is
unsubstituted
tetrahydrofuranyl) and are incorporated herewith.
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[0287] Thus, in embodiments, the A2A receptor antagonist is a compound of
formula:
N
Ni
\\N N NH2
R6 2
R6 I (II).
In formula (II), R6, R6-1 and R6-2 are independently hydrogen, halogen, =0,
=S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NIC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
[0288] In embodiments, the A2A receptor antagonist is a compound of formula:
_(
N
/N H2
-N
0
0
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[0289] In embodiments, the A2A receptor antagonist is a compound of formula:
_(
N..............."-\.õ/ .
, N
N
1
\
N---------.N.."'''.
) _______________ i NFI2
¨N
P
*
0o .
[0290] In embodiments, the A2A receptor antagonist is a compound of formula:
_(
4.Nk.,0,70
N, IN
\I
) _______________ / NNH2
N
0
d
0 .
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[0291] In embodiments, the method further includes administering a
therapeutically effective
amount of a PD-1 signaling pathway inhibitor. In embodiments, the A2A receptor
antagonist
and the PD-1 signaling pathway inhibitor are administered in a combined
synergistic amount. In
embodiments, the A2A receptor antagonist and the PD-1 signaling pathway
inhibitor are
administered simultaneously or sequentially. In embodiments, the A2A receptor
antagonist is
administered at a first time point and the PD-1 signaling pathway inhibitor is
administered at a
second time point, wherein the first time point precedes the second time
point. In embodiments,
the second time point is within less than about 120, 90, 60, 50, 40, 30, 20,
19, 18, 17, 16, 15, 14,
13, 12, 10, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days from the first time point.
In embodiments, the
second time point is within about 8, 10 or 12 days from the first time point.
In embodiments, the
PD-1 signaling pathway inhibitor is administered at a first time point and the
A2A receptor
antagonist is administered at a second time point, wherein the first time
point precedes the
second time point. In embodiments, the second time point is within less than
about 120, 90, 60,
50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 11, 9, 8, 7,6, 5, 4, 3, 2,
or 1 days from the first
time point. In embodiments, the second time point is within about 8, 10 or 12
days from the first
time point.
[0292] In embodiments, the A2A receptor antagonist is administered at an
amount of about 0.5
mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60
mg/kg, 70
mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 200 mg/kg or 300 mg/kg. In embodiments,
the A2A
receptor antagonist is administered at an amount of about 1 mg/kg. In
embodiments, the PD-1
signaling pathway inhibitor is administered at an amount of less than about
1,300 mg. In
embodiments, the PD-1 signaling pathway inhibitor is administered at an amount
of less than
about 1,200 mg. In embodiments, the cancer is selected from lung cancer,
bladder cancer,
melanoma, renal cell carcinoma, colon cancer, ovarian cancer, gastric cancer,
breast cancer, head
and neck carcinoma, prostate cancer and a hematologic malignancy.
METHODS OF ACTIVATING T CELLS
[0293] In one aspect, a method of activating a T cell is provided. The method
includes
contacting the T cell with an A2A receptor antagonist, wherein the A2A
receptor antagonist is a
compound of formula:
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RI
N
\N'NR2
R3
[0294] In formula (I), R1 is independently hydrogen, halogen, -C)03, -CN, -
S02C1, -S0111R9,
-S0,1NR9Rto, _NHNH2, _0NR9Rio, _NHc=(0)NHNH2, _NHc=(0)NR9Rio,
-NH-0-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9Rio, -0R9, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0n2R11,
-S0,2NRt1R12, NHNH2, 0NRtIR12, NHic _(0)NHNH2, NHic (0)NRit-K 12,
N(0)m2,
-NH-O-R", -C(0)R", _
K
C(0)-0R11, -C(0)NR11R12, -0R11, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CX'3, -CN, -
S02C1, -S0n3R13,
-S0,3NR13R14, _NHNH2, _coNR13R14, _NHc=(0)NHNH2, _NHc=(0)NR13Rt4 7 4\1(0)n137
4sTRI3R147 RI3 -C(0)R'3,
C(0)-0R13, -C(0)NR13R14, -0R13, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, Rut, Rit, R12, R13 and K-14
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
SO4H,
-SO2NH2, -NO2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. xa, Xb and Xc are independently -F, -Cl, -Br, or -I. n1, n2 and n3
are independently
an integer from 0 to 4. mt, m2 and m3 are independently an integer from 1 to
2. And vi, v2 and
v3 are independently an integer from 1 to 2.
[0295] The A2A receptor antagonist provided herein is the same A2A receptor
antagonist as
described above for aspects of treating cancer using an A2A receptor
antagonist and a PD-1
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signaling pathway inhibitor. Therefore, the definitions for substituents and
variables of formula
(I) and (II) are the same as described above (e.g., le is WA-substituted
furanyl; R1A is methyl; R2
is _NRI1R12; RH and R'2
are independently hydrogen; R3 is R4-substituted CI alkyl; R4 is R5-
substituted pyridinyl; R5 is R6-substituted 2 membered heteroalkyl; R6 is
unsubstituted
tetrahydrofuranyl) and are incorporated herewith.
[0296] Thus, in embodiments, the A2A receptor antagonist is a compound of
formula:
RI
\N NH2
R6 2
0
R" (II).
In formula (II), R6, R6.1 and R6.2 are independently hydrogen, halogen, =0,
=S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
[0297] In embodiments, the A2A receptor antagonist is a compound of formula:
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N
N
NH2
0
102981 In embodiments, the A2A receptor antagonist is a compound of formula:
ikk.zzvo
N /'NH2
-N
102991 In embodiments, the A2A receptor antagonist is a compound of formula:
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\N
Nai2
0
[0300] In embodiments, the method includes contacting the T cell with a PD-1
signaling
pathway inhibitor. In embodiments, the PD-1 signaling pathway inhibitor is an
antibody or a
small molecule. In embodiments, the T cell is an effector T cell or a natural
killer cell. In
embodiments, the T cell is an adenosine-suppressed T cell. "An adenosine-
suppressed T cell" is
an effector T cell or a natural killer cell bound to adenosine through its A2A
receptor, wherein
the adenosine is bound in an amount sufficient to inhibit expression and/or
secretion of immune
response activating cytokines (e.g., expression of IL-2, IFN-y or TNF). In
embodiments, the T
cell is a CD8 T cell. In embodiments, the CD8 T cell is a CD45RA-negative CD8
T cell. In
embodiments, the T cell is a CD4 T cell. In embodiments, the CD4 T cell is a
CD45RA-negative
CD4 T cell. In embodiments, the T cell is within a subject. In embodiments,
the subject is a
cancer subject. In embodiments, the cancer subject is an anti-PD-1 refractory
subject.
METHODS OF INHIBITING A2A RECEPTOR ACTIVITY
[0301] In one aspect, a method of inhibiting A2A receptor activity of a cell
is provided. The
method includes contacting the cell with an A2A receptor antagonist, wherein
the A2A receptor
antagonist is a compound of formula:
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RI
\N'NR2
R3
[0302] In formula (I), R1 is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -S0111R9,
_NHNH2, _0NR9Rio, _NHc=(0)NHNH2, _NHc="NR9Rio, _N(0).1,_NR9Rio,
-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR912.1o, -0R9, substituted or
unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0112R11,
_NHNH2, _NHc_(0)NHNH2, _NHc_(0)NRiiR12, _N(0)12,
K
NH-O-R", -C(0)1211, -C(0)-0R11, -C(0)NRIIR12, _0-11, substituted or
unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CX'3, -CN, -
S02C1, -S0n3R13,
-S0,3NR13R14, _NHNH27 _oNR13R14, _mic=(0)NHNH2, _Ntic=(0)NR13R14, _mow,
_NR13R14, _m-I-0-R13, -C(0)R'3,
C(0)-01213, -C(0)NR13R14, _oR13, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, Rio, R12, R13 and R'4
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
SO4H,
-SO2NH2, -NO2,
-NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. xa, Xb and Xc are independently -F, -Cl, -Br, or -I. ni, n2 and n3
are independently
an integer from 0 to 4. mi, m2 and m3 are independently an integer from 1 to
2. And vi, v2 and
V3 are independently an integer from 1 to 2.
[0303] The A2A receptor antagonist provided herein is the same A2A receptor
antagonist as
described above for aspects of treating cancer using an A2A receptor
antagonist and a PD-1
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signaling pathway inhibitor. Therefore, the definitions for substituents and
variables of formula
(I) and (II) are the same as described above (e.g., R1 is WA-substituted
furanyl; RIA is methyl; R2
is _NRI1R12; RH and R'2
are independently hydrogen; R3 is R4-substituted CI alkyl; R4 is R5-
substituted pyridinyl; R5 is R6-substituted 2 membered heteroalkyl; R6 is
unsubstituted
tetrahydrofuranyl) and are incorporated herewith.
[0304] Thus, in embodiments, the A2A receptor antagonist is a compound of
formula:
RI
\N NH2
R6 2
0
R" (II).
In formula (II), R6, R61 and R6=2 are independently hydrogen, halogen, =0, =S,
-CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
[0305] In embodiments, the A2A receptor antagonist is a compound of formula:
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N
N
NH2
0
103061 In embodiments, the A2A receptor antagonist is a compound of formula:
ikk.zzvo
N /'NH2
-N
103071 In embodiments, the A2A receptor antagonist is a compound of formula:
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\N
Nai2
0
[0308] In embodiments, the contacting includes binding the A2A receptor
antagonist to an
A2A receptor of the cell. In embodiments, the cell is a T cell. In
embodiments, the T cell is an
effector T cell or a natural killer cell. In embodiments, T cell is a CD8 T
cell. In embodiments,
the CD8 T cell is a CD45RA-negative CD8 Tcell. In embodiments, the T cell is a
CD4 Tcell. In
embodiments, the CD4 T cell is a CD45RA-negative CD4 Tcell. In embodiments,
the T cell is
within a subject. In embodiments, the subject is a cancer subject. In
embodiments, the cancer
subject is an anti-PD-1 refractory subject.
METHODS OF INCREASING ANTI-TUMOR RESPONSE
[0309] In one aspect, a method of increasing an anti-tumor immune response in
a subject in
need thereof is provided. The method includes administering to the subject a
therapeutically
effective amount of an adenosine-A2A (A2A) receptor antagonist and a
programmed cell death
protein 1 (PD-1) signaling pathway inhibitor.
[0310] In another aspect, a method of increasing an anti-tumor immune response
in a subject
in need thereof is provided. The method includes administering to the subject
a therapeutically
effective amount of an adenosine-A2A (A2A) receptor antagonist, wherein the
A2A receptor
antagonist is a compound of formula:
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RI
\N'NR2
R3
[0311] In formula (I), R1 is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -S0111R9,
-S0,1NR9Rm, _NHNH2, _0NR9R1o, _NHc=(0)NHNH2, _NHc=(0)NR9R1o, _N(0).1,_NR9R1o,
-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR912.1o, -0R9, substituted or
unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0112R11,
-S0,2NRiiRt2, _NHNH2, _0NRiiRt2, _NHc_(0)NHNH2, _NHc_(0)NRItR12, _N(0)12,
K
NH-O-R", -C(0)1211, -C(0)-0R11, -C(0)NRIIR12, _0-11, substituted or
unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CX'3, -CN, -
S02C1, -S0n3R13,
-S0,3NR13R14, _NHNH2, _0NR13R14, _NHc=(0)NHNH2, _NHc=(0)NR13R14, _mow,
_NR13R14, _m-I-0-R13, -C(0)R'3,
C(0)-01213, -C(0)NR13R14, _oR13, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, Ru", R12, RI3 and R'4
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
SO4H,
-SO2NH2, -NO2,
-NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. xa, Xb and Xc are independently -F, -Cl, -Br, or -I. ni, n2 and n3
are independently
an integer from 0 to 4. m", m2 and m3 are independently an integer from 1 to
2. And v", v2 and
V3 are independently an integer from 1 to 2.
[0312] The A2A receptor antagonist and the PD-1 signaling pathway inhibitor
provided herein
are the same as described above for aspects of treating cancer using an A2A
receptor antagonist
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and a PD-1 signaling pathway inhibitor. Therefore, the definitions for
substituents and variables
of folinula (I) and (II) are the same as described above (e.g., le is WA-
substituted furanyl; WA is
methyl; R2 is _NRI1R12; RH. and R'2
are independently hydrogen; R3 is R4-substituted C1 alkyl;
R4 is R5-substituted pyridinyl; R5 is R6-substituted 2 membered heteroalkyl;
R6 is unsubstituted
tetrahydrofuranyl) and are incorporated herewith.
[0313] In embodiments, the A2A receptor antagonist is a compound of formula:
RI
N
\N NH2
R6 2
R" (II).
In formula (II), R6, R6.1 and R6.2 are independently hydrogen, halogen, =0,
=S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
[0314] In embodiments, the A2A receptor antagonist is a compound of formula:
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N
N
NH2
0
103151 In embodiments, the A2A receptor antagonist is a compound of formula:
ikk.zzvo
N /'NH2
-N
103161 In embodiments, the A2A receptor antagonist is a compound of formula:
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\N
Nai2
0
[0317] In embodiments, the method includes administering a therapeutically
effective amount
of a PD-1 signaling pathway inhibitor. In embodiments, the PD-1 signaling
pathway inhibitor is
a PD-Li antagonist. In embodiments, the PD-Li antagonist is a small molecule
or an antibody.
METHODS OF INCREASING CD8-POSITIVE CELL NUMBERS
[0318] In one aspect, a method of increasing the amount of CD8-positive cells
relative to the
amount of regulatory T cells in a subject in need thereof is provided. The
method includes
administering to the subject a therapeutically effective amount of an
adenosine-A2A (A2A)
receptor antagonist and a programmed cell death protein 1 (PD-1) signaling
pathway inhibitor.
[0319] In one aspect, a method of increasing the amount of CD8-positive cells
relative to the
amount of regulatory T cells in a subject in need thereof is provided. The
method includes
administering to the subject a therapeutically effective amount of an
adenosine-A2A (A2A)
receptor antagonist, wherein the A2A receptor antagonist is a compound of
formula:
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RI
\N'NR2
R3
[0320] In formula (I), R1 is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -S00R9,
-S0,1NR9Rio, _NHNH2, _0NR9Rio, _NHc=(0)NHNH2, _NHc="NR9Rio, _N(0).1,_NR9Rio,
-NH-0-R9, -C(0)R9, -C(0)-0R9, -C(0)NR912.1o, -0R9, substituted or
unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0112R11,
-S0,2NRiiRt2, _NHNH2, _NHc_(0)NHNH2, _NHc_oz,NRiiR12, _N(0)12,
K
NH-O-R", -C(0)1211, -C(0)-0R11, -C(0)NRIIR12, _0-11, substituted or
unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CX'3, -CN, -
S02C1, -S0n3R13,
-S0,3NR13R14, _NHNH27 _oNR13R14, _mic=(0)NHNH2, _NHc="NR13R14, _mow,
_NR13R14, _m-I-0-R13, -C(0)R'3,
C(0)-01213, -C(0)NR13R14, _oR13, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, Rio, R12, R13 and R'4
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
SO4H,
-SO2NH2, -NO2,
-NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. xa, Xb and Xc are independently -F, -Cl, -Br, or -I. ni, n2 and n3
are independently
an integer from 0 to 4. mi, m2 and m3 are independently an integer from 1 to
2. And vi, v2 and
V3 are independently an integer from 1 to 2.
[0321] The A2A receptor antagonist and the PD-1 signaling pathway inhibitor
provided herein
are the same as described above for aspects of treating cancer using an A2A
receptor antagonist
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and a PD-1 signaling pathway inhibitor. Therefore, the definitions for
substituents and variables
of folinula (I) and (II) are the same as described above (e.g., le is WA-
substituted furanyl; WA is
methyl; R2 is _NRI1R12; RH. and R'2
are independently hydrogen; R3 is R4-substituted C1 alkyl;
R4 is R5-substituted pyridinyl; R5 is R6-substituted 2 membered heteroalkyl;
R6 is unsubstituted
tetrahydrofuranyl) and are incorporated herewith.
[0322] In embodiments, the A2A receptor antagonist is a compound of formula:
RI
N
\N NH2
R6 2
R" (II).
In formula (II), R6, R6.1 and R6.2 are independently hydrogen, halogen, =0,
=S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
[0323] In embodiments, the A2A receptor antagonist is a compound of formula:
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N
N
NH2
0
103241 In embodiments, the A2A receptor antagonist is a compound of formula:
ikk.zzvo
N /'NH2
-N
103251 In embodiments, the A2A receptor antagonist is a compound of formula:
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\N
Nai2
0
[0326] In embodiments, the method includes administering a therapeutically
effective amount
of a PD-1 signaling pathway inhibitor. In embodiments, the PD-1 signaling
pathway inhibitor is
a PD-Li antagonist. In embodiments, the PD-Li antagonist is a small molecule
or an antibody.
METHODS OF DECREASING TUMOR VOLUME
[0327] In one aspect, a method of decreasing tumor volume in a subject in need
thereof is
provided. The method includes administering to the subject a therapeutically
effective amount of
an adenosine-A2A (A2A) receptor antagonist and a programmed cell death protein
1 (PD-1)
signaling pathway inhibitor.
[0328] In one aspect, a method of decreasing tumor volume in a subject in need
thereof is
provided. The method includes administering to the subject a therapeutically
effective amount of
an adenosine-A2A (A2A) receptor antagonist, wherein the A2A receptor
antagonist is a
compound of formula:
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RI
\N'NR2
R3
[0329] In formula (I), R1 is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -S00R9,
-S0,1NR9Rio, _NHNH2, _0NR9Rio, _NHc=(0)NHNH2, _NHc="NR9Rio, _N(0).1,_NR9Rio,
-NH-0-R9, -C(0)R9, -C(0)-0R9, -C(0)NR912.1o, -0R9, substituted or
unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0112R11,
-S0,2NRiiRt2, _NHNH2, _NHc_(0)NHNH2, _NHc_oz,NRiiR12, _N(0)12,
K
NH-O-R", -C(0)1211, -C(0)-0R11, -C(0)NRIIR12, _0-11, substituted or
unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CX'3, -CN, -
S02C1, -S0n3R13,
-S0,3NR13R14, _NHNH27 _oNR13R14, _mic=(0)NHNH2, _NHc="NR13R14, _mow,
_NR13R14, _m-I-0-R13, -C(0)R'3,
C(0)-01213, -C(0)NR13R14, _oR13, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, Rio, R12, R13 and R'4
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
SO4H,
-SO2NH2, -NO2,
-NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. xa, Xb and Xc are independently -F, -Cl, -Br, or -I. ni, n2 and n3
are independently
an integer from 0 to 4. mi, m2 and m3 are independently an integer from 1 to
2. And vi, v2 and
V3 are independently an integer from 1 to 2.
[0330] The A2A receptor antagonist and the PD-1 signaling pathway inhibitor
provided herein
are the same as described above for aspects of treating cancer using an A2A
receptor antagonist
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and a PD-1 signaling pathway inhibitor. Therefore, the definitions for
substituents and variables
of folinula (I) and (II) are the same as described above (e.g., le is WA-
substituted furanyl; WA is
methyl; R2 is _NRI1R12; RH. and R'2
are independently hydrogen; R3 is R4-substituted C1 alkyl;
R4 is R5-substituted pyridinyl; R5 is R6-substituted 2 membered heteroalkyl;
R6 is unsubstituted
tetrahydrofuranyl) and are incorporated herewith.
[0331] In embodiments, the A2A receptor antagonist is a compound of formula:
RI
N
\N NH2
R6 2
R" (II).
In formula (II), R6, R6.1 and R6.2 are independently hydrogen, halogen, =0,
=S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
[0332] In embodiments, the A2A receptor antagonist is a compound of formula:
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N
N
NH2
0
103331 In embodiments, the A2A receptor antagonist is a compound of formula:
ikk.zzvo
N /'NH2
-N
103341 In embodiments, the A2A receptor antagonist is a compound of formula:
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\N
Nai2
0
[0335] In embodiments, the method includes administering a therapeutically
effective amount
of a PD-1 signaling pathway inhibitor. In embodiments, the PD-1 signaling
pathway inhibitor is
a PD-Li antagonist. In embodiments, the PD-Li antagonist is a small molecule
or an antibody.
METHODS OF ENHANCING ANTI-TUMOR MEMORY
[0336] In one aspect, a method of enhancing anti-tumor immune memory in a
subject in need
thereof is provided. The method includes administering to the subject a
therapeutically effective
amount of an adenosine-A2A (A2A) receptor antagonist and a programmed cell
death protein 1
(PD-1) signaling pathway inhibitor.
[0337] In one aspect, a method of enhancing anti-tumor immune memory in a
subject in need
thereof is provided. The method includes administering to the subject a
therapeutically effective
amount of an adenosine-A2A (A2A) receptor antagonist, wherein the A2A receptor
antagonist is
a compound of formula:
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RI
\N'NR2
R3
[0338] In formula (I), R1 is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -S00R9,
-S0,1NR9Rio, _NHNH2, _0NR9Rio, _NHc=(0)NHNH2, _NHc="NR9Rio, _N(0).1,_NR9Rio,
-NH-0-R9, -C(0)R9, -C(0)-0R9, -C(0)NR912.1o, -0R9, substituted or
unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0112R11,
-S0,2NRiiRt2, _NHNH2, _NHc_(0)NHNH2, _NHc_oz,NRiiR12, _N(0)12,
K
NH-O-R", -C(0)1211, -C(0)-0R11, -C(0)NRIIR12, _0-11, substituted or
unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CX'3, -CN, -
S02C1, -S0n3R13,
-S0,3NR13R14, _NHNH27 _oNR13R14, _mic=(0)NHNH2, _NHc="NR13R14, _mow,
_NR13R14, _m-I-0-R13, -C(0)R'3,
C(0)-01213, -C(0)NR13R14, _oR13, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, Rio, R12, R13 and R'4
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -
SO4H,
-SO2NH2, -NO2,
-NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. xa, Xb and Xc are independently -F, -Cl, -Br, or -I. ni, n2 and n3
are independently
an integer from 0 to 4. mi, m2 and m3 are independently an integer from 1 to
2. And vi, v2 and
V3 are independently an integer from 1 to 2.
[0339] The A2A receptor antagonist and the PD-1 signaling pathway inhibitor
provided herein
are the same as described above for aspects of treating cancer using an A2A
receptor antagonist
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and a PD-1 signaling pathway inhibitor. Therefore, the definitions for
substituents and variables
of folinula (I) and (II) are the same as described above (e.g., RI- is R1A-
substituted furanyl; RA is
methyl; R2 is _NRI1R12; RH. and R'2
are independently hydrogen; R3 is R4-substituted C1 alkyl;
R4 is R5-substituted pyridinyl; R5 is R6-substituted 2 membered heteroalkyl;
R6 is unsubstituted
tetrahydrofuranyl) and are incorporated herewith.
[0340] In embodiments, the A2A receptor antagonist is a compound of formula:
RI
N
\N NH2
R6 2
R" (II).
In formula (II), R6, R61 and R6=2 are independently hydrogen, halogen, =0, =S,
-CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
[0341] In embodiments, the A2A receptor antagonist is a compound of formula:
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N
N
NH2
0
103421 In embodiments, the A2A receptor antagonist is a compound of formula:
ikk.zzvo
N /'NH2
-N
103431 In embodiments, the A2A receptor antagonist is a compound of formula:
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N
Nai2
-N
0
[0344] In embodiments, the method includes administering a therapeutically
effective amount
of a PD-1 signaling pathway inhibitor. In embodiments, the PD-1 signaling
pathway inhibitor is
a PD-Li antagonist. In embodiments, the PD-Li antagonist is a small molecule
or an antibody.
[0345] In one aspect, a method of increasing global immune activation in a
subject in need
thereof is provided. The method includes administering to said subject a
therapeutically
effective amount of an adenosine-A2A (A2A) receptor antagonist, wherein the
A2A receptor
antagonist is a compound of formula:
RI
Na
NNR
R3 (0,
[0346] In formula (I), is independently hydrogen, halogen, -CV3, -CN, -
S02C1, -S00R9,
-SOvINR9Rio, _NHNH2, _0NR9Rio, _NHc=(0)NHNH2, _mic=(0)NR9Rio, _N(0).1,_NR9Rio,
-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9R1 -0R9, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
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unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0n2R11,
-S0,2NRitRu, NHNH2, 0NRI IR12, NHC=(0)NHNH2, -NHC=(0)NR1iRu, _N(0),n2,
_NRItRu, -NH-O-R", -C(0)R", -C(0)-OR", _c(0)NRitRu, -OR",
substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl. R3 is independently hydrogen,
halogen, -CXc3, -CN,
-S02C1, -S0n3R", -S0,3NRi3R14, NTNH2, 0NR t3R14, NHic (0)NHNH2,
-NHC-(0)NR13Ri4,_N(0).3, _NR13,--K 14, - NH-0-Ru, -C(0)R13, -C(0)-ORH, -
C(0)NR13R14,
-0R13, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl. R9, RI , Rn,
Ru, R'3
and R" are
independently hydrogen, halogen, =0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -
CONH2,
-OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2, -NO2, -NH2, -NHNH2, -ONH2, -
NHC=(0)NHNH,
substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl. Xa, Xb and X'
are independently
-F, -Cl, -Br, or -I. ni, n2 and n3 are independently an integer from 0 to 4.
ml, m2 and m3 are
independently an integer from 1 to 2. v1, v2 and v3 are independently an
integer from 1 to 2.
[0347] In embodiments, the A2A receptor antagonist is a compound of formula:
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N
N
NH2
0
103481 In embodiments, the A2A receptor antagonist is a compound of formula:
ikk.zzvo
N /'NH2
-N
103491 In embodiments, the A2A receptor antagonist is a compound of formula:
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\N
Nai2
0
103501 In embodiments, the method further includes administering a
therapeutically effective
amount of a PD-1 signaling pathway inhibitor. In embodiments, the PD-1
signaling pathway
inhibitor is a PD-Li antagonist. In embodiments, the PD-Li antagonist is a
small molecule or an
antibody. In embodiments, the method includes activating a CD4 T cell in the
subject. In
embodiments, the CD4 T cell is a memory T cell. In embodments, CD4 T cell is
an effector T
cell.
[0351] In embodiments, the relative amount of CD45RA-negative CD4 T cells in
the subject is
increased. In embodiments, the relative amount of CD4 T cells in the subject
is increased.
Where the relative amount of CD4 T cells in the subject is increased the
amount of CD4 T cells
in the subject can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than
that in a
control. In certain instances, the increase is 1.5-fold, 2-fold, 3-fold, 4-
fold, 5-fold, 10-fold, or
more in comparison to a control. In embodiments, the relative amount of memory
T cells in the
subject is increased. Where the relative amount of memory T cells in the
subject is increased the
amount of memory T cells in the subject can be 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%,
90% or more than that in a control. In certain instances, the increase is 1.5-
fold, 2-fold, 3-fold,
4-fold, 5-fold, 10-fold, or more in comparison to a control. In embodiments,
the relative amount
of effector T cells in the subject is increased. Where the relative amount of
effector T cells in the
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subject is increased the amount of effector T cells in the subject can be 10%,
20%, 30%, 40%,
50%, 60%, 70%, 80%, 90% or more than that in a control. In certain instances,
the increase is
1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in comparison to a
control. In
embodiments, the method includes increasing the number of PD-1 positive cells
in the subject.
Where the number of PD-1 positive cells in the subject is increased the amount
of PD-1 positive
cells in the subject can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
more than that
in a control. In certain instances, the increase is 1.5-fold, 2-fold, 3-fold,
4-fold, 5-fold, 10-fold,
or more in comparison to a control.
[0352] In embodiments, the method includes activating a CD8 T cell in the
subject. In
embodiments, the relative amount of CD8 T cells in the subject is increased.
In embodiments,
the relative frequency of TCR recombination is increased. Where the relative
frequency of TCR
recombination is increased the amounts of TCR recombination events can be 10%,
20%, 30%,
40%, 50%, 60%, 70%, 80%, 90% or more than that in a control. In certain
instances, the
increase is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in
comparison to a control.
Where the frequency of TCR recombination is increased, the T cell receptor
repertoire (the
number of T cells recognizing antigens that are chemically different from each
other) is
increased. Thus, the methods provided herein may increase the diversity of T
cell clones in the
subject.
[0353] In embodiments, the subject is an anti-PD-1 refractory subject.
[0354] For the methods provided herein the A2A receptor antagonist may be
administered at
an amount of about 100 mg BID. In embodiments, the A2A receptor antagonist is
administered
for 28 consecutive days. In embodiments, the A2A receptor antagonist is
administered for 14
consecutive days. In embodiments, the PD-1 signaling pathway inhibitor is
administered at an
amount of about 840 mg. In embodiments, the PD-1 signaling pathway inhibitor
is administered
for 28 consecutive days. In embodiments, the PD-1 signaling pathway inhibitor
is administered
for 14 consecutive days. In furher embodiments, the A2A receptor antagonist
and the PD-1
signaling pathway inhibitor are administered on the same day.
103551 In embodiments, the A2A receptor antagonist is administered at a first
time point and
sthe PD-1 signaling pathway inhibitor is administered at a second time point,
wherein the first
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time point precedes the second time point. In embodiments, the second time
point is within less
than about 120, 90, 60, 50, 40, 30, 28, 20, 19, 18, 17, 16, 15, 14, 13, 12,
11, 10, 9, 8, 7, 6, 5, 4, 3,
2 or 1 days from said first time point. In embodiments, the second time point
is within less than
about 120 days. In embodiments, the second time point is within less than
about 90 days. In
embodiments, the second time point is within less than about 60 days. In
embodiments, the
second time point is within less than about 50 days. In embodiments, the
second time point is
within less than about 40 days. In embodiments, the second time point is
within less than about
30 days. In embodiments, the second time point is within less than about 28
days. In
embodiments, the second time point is within less than about 20 days. In
embodiments, the
second time point is within less than about 19 days. In embodiments, the
second time point is
within less than about 18 days. In embodiments, the second time point is
within less than about
17 days. In embodiments, the second time point is within less than about 16
days. In
embodiments, the second time point is within less than about 15 days. In
embodiments, the
second time point is within less than about 14 days. In embodiments, the
second time point is
within less than about 13 days. In embodiments, the second time point is
within less than about
12 days. In embodiments, the second time point is within less than about 11
days. In
embodiments, the second time point is within less than about 10 days. In
embodiments, the
second time point is within less than about 9 days. In embodiments, the second
time point is
within less than about 8 days. In embodiments, the second time point is within
less than about 7
days. In embodiments, the second time point is within less than about 6 days.
In embodiments,
the second time point is within less than about 5 days. In embodiments, the
second time point is
within less than about 4 days. In embodiments, the second time point is within
less than about 3
days. In embodiments, the second time point is within less than about 2 days.
In embodiments,
the second time point is within less than about 2 days. In embodiments, the
second time point is
within less than about 1 day.
103561 In embodiments, the second time point is within about 14 or 28 days
from the first time
point. In embodiments, the second time point is within about 14 days from the
first time point.
In embodiments, the second time point is within about 28 days from the first
time point.
[0357] The methods provided herein including embodiments thereof may include
activating a
T cell in the subject. The methods provided herein including embodiments
thereof may include
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activating a CD4T cell in the subject. In embodiments, the CD4 T cell is a
memory T cell. In
embodiments, the CD4 T cell is an effector T cell. In embodiments, the CD4 T
cell is a
CD45RA-negative CD4 T cell. In embodiments, the relative amount of a CD4 T
cell is
increased in the subject. In embodiments, the relative amount of an effector T
cell is increased in
the subject. In embodiments, the relative amount of a CD45RA-negative CD4 T
cell is increased
in the subject.
[0358] The methods provided herein including embodiments thereof may include
inhibiting
A2A receptor activity of a cell in the subject. The methods provided herein
including
embodiments thereof may include increasing an anti-tumor immune response in a
subject. The
methods provided herein including embodiments thereof may include increasing
the amount of
CD8-positive cells relative to the amount of regulatory T cells in the
subject. The methods
provided herein including embodiments thereof may include enhancing anti-tumor
immune
memory in the subject. The methods provided herein including embodiments
thereof may
include enhancing anti-tumor immune memory in the subject. The methods
provided herein
including embodiments thereof may include increasing global immune activation
in the subject.
PHARMACEUTICAL COMPOSITIONS
[0359] Provided herein are pharmaceutical compositions including an A2A
receptor
antagonist, a PD-1 signaling pathway inhibitor and a pharmaceutically
acceptable excipient. The
provided compositions are, inter al/a, suitable for formulation and
administration in vitro or in
vivo. Suitable carriers and excipients and their formulations are described in
Remington: The
Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott
Williams &
Wilkins (2005). By pharmaceutically acceptable carrier is meant a material
that is not
biologically or otherwise undesirable, i.e., the material is administered to a
subject without
causing undesirable biological effects or interacting in a deleterious manner
with the other
components of the pharmaceutical composition in which it is contained. If
administered to a
subject, the carrier is optionally selected to minimize degradation of the
active ingredient and to
minimize adverse side effects in the subject.
[0360] Compositions can be administered for therapeutic or prophylactic
treatments. In
therapeutic applications, compositions are administered to a patient suffering
from a disease
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(e.g., cancer) in a "therapeutically effective dose." Amounts effective for
this use will depend
upon the severity of the disease and the general state of the patient's
health. Single or multiple
administrations of the compositions may be administered depending on the
dosage and frequency
as required and tolerated by the patient.
[0361] Pharmaceutical compositions provided by the present invention include
compositions
wherein the active ingredient (e.g. compositions described herein, including
embodiments or
examples) is contained in a therapeutically effective amount, i.e., in an
amount effective to
achieve its intended purpose. The actual amount effective for a particular
application will
depend, inter al/a, on the condition being treated. When administered in
methods to treat a
disease, the compounds and antibodies described herein will contain an amount
of active
ingredient effective to achieve the desired result, e.g., modulating the
activity of a target
molecule, and/or reducing, eliminating, or slowing the progression of disease
symptoms.
DeteHnination of a therapeutically effective amount of a compound of the
invention is well
within the capabilities of those skilled in the art, especially in light of
the detailed disclosure
herein.
[0362] Provided compositions can include a single agent or more than one
agent. The
compositions for administration will commonly include an agent as described
herein dissolved in
a pharmaceutically acceptable carrier, preferably an aqueous carrier, A
variety of aqueous
carriers can be used, e.g., buffered saline and the like. These solutions are
sterile and generally
free of undesirable matter. These compositions may be sterilized by
conventional, well known
sterilization techniques. The compositions may contain pharmaceutically
acceptable auxiliary
substances as required to approximate physiological conditions such as pH
adjusting and
buffering agents, toxicity adjusting agents and the like, for example, sodium
acetate, sodium
chloride, potassium chloride, calcium chloride, sodium lactate and the like.
The concentration of
active agent in these formulations can vary widely, and will be selected
primarily based on fluid
volumes, viscosities, body weight and the like in accordance with the
particular mode of
administration selected and the subject's needs.
[0363] Solutions of the active compounds as free base or pharmacologically
acceptable salt
can be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose.
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Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and
mixtures thereof
and in oils. Under ordinary conditions of storage and use, these preparations
can contain a
preservative to prevent the growth of microorganisms.
[0364] Pharmaceutical compositions can be delivered via intranasal or
inhalable solutions or
sprays, aerosols or inhalants. Nasal solutions can be aqueous solutions
designed to be
administered to the nasal passages in drops or sprays. Nasal solutions can be
prepared so that
they are similar in many respects to nasal secretions. Thus, the aqueous nasal
solutions usually
are isotonic and slightly buffered to maintain a pH of 5.5 to 6.5. In
addition, antimicrobial
preservatives, similar to those used in ophthalmic preparations and
appropriate drug stabilizers, if
required, may be included in the formulation. Various commercial nasal
preparations are known
and can include, for example, antibiotics and antihistamines.
[0365] Oral formulations can include excipients as, for example,
pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium
carbonate and the like. These compositions take the form of solutions,
suspensions, tablets, pills,
capsules, sustained release formulations or powders. In some embodiments, oral
pharmaceutical
compositions will comprise an inert diluent or assimilable edible carrier, or
they may be enclosed
in hard or soft shell gelatin capsule, or they may be compressed into tablets,
or they may be
incorporated directly with the food of the diet. For oral therapeutic
administration, the active
compounds may be incorporated with excipients and used in the form of
ingestible tablets,
buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and
the like. Such
compositions and preparations should contain at least 0.1% of active compound.
The percentage
of the compositions and preparations may, of course, be varied and may
conveniently be between
about 2 to about 75% of the weight of the unit, or preferably between 25-60%.
The amount of
active compounds in such compositions is such that a suitable dosage can be
obtained.
[0366] For parenteral administration in an aqueous solution, for example, the
solution should
be suitably buffered and the liquid diluent first rendered isotonic with
sufficient saline or
glucose. Aqueous solutions, in particular, sterile aqueous media, are
especially suitable for
intravenous, intramuscular, subcutaneous and intraperitoneal administration.
For example, one
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dosage could be dissolved in 1 ml of isotonic NaCl solution and either added
to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion.
[0367] Sterile injectable solutions can be prepared by incorporating the
active compounds or
constructs in the required amount in the appropriate solvent followed by
filtered sterilization.
Generally, dispersions are prepared by incorporating the various sterilized
active ingredients into
a sterile vehicle which contains the basic dispersion medium. Vacuum-drying
and freeze-drying
techniques, which yield a powder of the active ingredient plus any additional
desired ingredients,
can be used to prepare sterile powders for reconstitution of sterile
injectable solutions. The
preparation of more, or highly, concentrated solutions for direct injection is
also contemplated.
DMSO can be used as solvent for extremely rapid penetration, delivering high
concentrations of
the active agents to a small area.
[0368] The formulations of compounds can be presented in unit-dose or multi-
dose sealed
containers, such as ampules and vials. Thus, the composition can be in unit
dosage form. In
such form the preparation is subdivided into unit doses containing appropriate
quantities of the
active component. Thus, the compositions can be administered in a variety of
unit dosage forms
depending upon the method of administration. For example, unit dosage forms
suitable for oral
administration include, but are not limited to, powder, tablets, pills,
capsules and lozenges.
[0369] The dosage and frequency (single or multiple doses) administered to a
mammal can
vary depending upon a variety of factors, for example, whether the mammal
suffers from another
disease, and its route of administration; size, age, sex, health, body weight,
body mass index, and
diet of the recipient; nature and extent of symptoms of the disease being
treated (e.g. symptoms
of cancer and severity of such symptoms), kind of concurrent treatment,
complications from the
disease being treated or other health-related problems. Other therapeutic
regimens or agents can
be used in conjunction with the methods and compounds of the invention.
Adjustment and
manipulation of established dosages (e.g., frequency and duration) are well
within the ability of
those skilled in the art.
[0370] For any composition (e.g., the compounds and antibodies provided)
described herein,
the therapeutically effective amount can be initially determined from cell
culture assays. Target
concentrations will be those concentrations of active compound(s) that are
capable of achieving
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the methods described herein, as measured using the methods described herein
or known in the
art. As is well known in the art, effective amounts for use in humans can also
be determined
from animal models. For example, a dose for humans can be formulated to
achieve a
concentration that has been found to be effective in animals. The dosage in
humans can be
adjusted by monitoring effectiveness and adjusting the dosage upwards or
downwards, as
described above. Adjusting the dose to achieve maximal efficacy in humans
based on the
methods described above and other methods is well within the capabilities of
the ordinarily
skilled artisan.
[0371] Dosages may be varied depending upon the requirements of the patient
and the
compound being employed. The dose administered to a patient, in the context of
the present
invention should be sufficient to affect a beneficial therapeutic response in
the patient over time.
The size of the dose also will be determined by the existence, nature, and
extent of any adverse
side-effects. Determination of the proper dosage for a particular situation is
within the skill of
the practitioner. Generally, treatment is initiated with smaller dosages which
are less than the
optimum dose of the compound. Thereafter, the dosage is increased by small
increments until
the optimum effect under circumstances is reached.
[0372] Dosage amounts and intervals can be adjusted individually to provide
levels of the
administered compound effective for the particular clinical indication being
treated. This will
provide a therapeutic regimen that is commensurate with the severity of the
individual's disease
state.
[0373] Utilizing the teachings provided herein, an effective prophylactic or
therapeutic
treatment regimen can be planned that does not cause substantial toxicity and
yet is effective to
treat the clinical symptoms demonstrated by the particular patient. This
planning should involve
the careful choice of active compound by considering factors such as compound
potency, relative
bioavailability, patient body weight, presence and severity of adverse side
effects, preferred
[0374] "Pharmaceutically acceptable excipient" and "pharmaceutically
acceptable carrier"
refer to a substance that aids the administration of an active agent to and
absorption by a subject
and can be included in the compositions of the present invention without
causing a significant
adverse toxicological effect on the patient. Non-limiting examples of
pharmaceutically
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acceptable excipients include water, NaCl, normal saline solutions, lactated
Ringer's, normal
sucrose, normal glucose, binders, fillers, disintegrants, lubricants,
coatings, sweeteners, flavors,
salt solutions (such as Ringer's solution), alcohols, oils, gelatins,
carbohydrates such as lactose,
amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl
pyrrolidine, and colors,
and the like. Such preparations can be sterilized and, if desired, mixed with
auxiliary agents
such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers,
salts for influencing
osmotic pressure, buffers, coloring, and/or aromatic substances and the like
that do not
deleteriously react with the compounds of the invention. One of skill in the
art will recognize
that other pharmaceutical excipients are useful in the present invention.
[0375] The term "pharmaceutically acceptable salt" refers to salts derived
from a variety of
organic and inorganic counter ions well known in the art and include, by way
of example only,
sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the
like; and
when the molecule contains a basic functionality, salts of organic or
inorganic acids, such as
hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and
the like.
[0376] The term "preparation" is intended to include the formulation of the
active compound
with encapsulating material as a carrier providing a capsule in which the
active component with
or without other carriers, is surrounded by a carrier, which is thus in
association with it.
Similarly, cachets and lozenges are included. Tablets, powders, capsules,
pills, cachets, and
lozenges can be used as solid dosage forms suitable for oral administration.
[0377] In one aspect, a pharmaceutical composition including an A2A receptor
antagonist, a
PD-1 signaling pathway inhibitor and a pharmaceutically acceptable excipient
is provided.
[0378] In embodiments, the A2A receptor antagonist is a compound of formula:
RI
\NR2
R3
[0379] In formula (I), le is independently hydrogen, halogen, -CXa3, -CN, -
S02C1, -50111R9,
_NHNH2, _0NR9Rio, _NHC=(0)NHNH2, ¨NHC=(0)NR9Rio, _N(0).",_NR9Rio,
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-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9R ot -0R9, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -
S0.2R11,
-S0,2NR11R12, rµarsi-H2, 0NRI1R12, NHic _(0)NHN-H2, Nuc _(0)NRitR12, _N(0)m2,
_NRiiR12, -NH-O-R", -C(0)R", -C(0)-OR", -C(0)NRI1R12,
11, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R3 is independently hydrogen, halogen, -CXC3, -CN, -
S02C1, -S0n3R13,
-S0v3NR13R14, _NHNH2, -0NRI3R14, _NHC-(0)NHNH2, -NHC-(0)NRBRI4, _N(0).3,
_NRnR14, _c(c)Rn, -C(0)-0R13, -C(0)NRnRi4, _0-c 13, substituted
or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or
unsubstituted heteroaryl. R9, Rio, Rn, R12, R13 and R'4
are independently hydrogen, halogen,
=0, =S, -CF3, -CN, -CC13, -COOH, -CH2COOH, -CONF12, -OH, -SH, -S02C1, -S03H, -
SO4H,
-S021\1H2, -NO2, -NHz, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or
unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl. Xa, Xb and X' are independently -F, -Cl, -Br, or -I. ni, nz and n3
are independently
an integer from 0 to 4. ml, m2 and m3 are independently an integer from 1 to
2. And VI, vz and
V3 are independently an integer from 1 to 2.
[0380] The A2A receptor antagonist and the PD-1 signaling pathway inhibitor
provided herein
are the same as described above for aspects of treating cancer using an A2A
receptor antagonist
and a PD-1 signaling pathway inhibitor. Therefore, the definitions for
substituents and variables
of fonnula (I) and (II) are the same as described above (e.g., RI is RIA-
substituted furanyl; RIA is
methyl; R2 is _NRiiR12, Rn and R'2
are independently hydrogen; R3 is R4-substituted CI alkyl;
R4 is R5-substituted pyridinyl; R5 is R6-substituted 2 membered heteroalkyl;
R6 is unsubstituted
tetrahydrofuranyl) and are incorporated herewith.
[0381] In embodiments, the A2A receptor antagonist is a compound of formula:
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/
NH2
R6 2
-R6
R6 (II).
In formula (II), R6, R6.1 and R6.2 are independently hydrogen, halogen, =0,
=S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NH2, -NTINH2, -ONH2, -NHC=(0)NHINH2, substituted or unsubstituted
alkyl, substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
[0382] In embodiments, the A2A receptor antagonist is a compound of formula:
N
NNNH2
0
0
[0383] In embodiments, the A2A receptor antagonist is a compound of formula:
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N/
N
NH2
9
[0384] In embodiments, the A2A receptor antagonist is a compound of folinula:
N/
N
\N
-N
0
[0385] In embodiments, the PD-1 signaling pathway inhibitor is a programmed
death-ligand 1
(PD-L1) antagonist or a PD-1 antagonist. In embodiments, the programmed death-
ligand 1 (PD-
L1) antagonist is an antibody or a small molecule. In embodiments, the PD-Li
antagonist is an
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antibody. In embodiments, the antibody is atezolizumab. In embodiments, the PD-
1 antagonist
is an antibody or a small molecule. In embodiments, the A2A receptor
antagonist and the PD-1
signaling pathway inhibitor are present in a combined synergistic amount,
wherein the combined
synergistic amount is effective to treat cancer in a subject in need thereof.
[0386] In embodiments, the pharmaceutical composition is in oral dosage form.
In
embodiments, the adenosine-A2A (A2A) receptor antagonist(e.g, CPI-444) is
presented as size
0-elongated hydroxypropyl methylcellulose (HPMC) capsules containing adenosine-
A2A (A2A)
receptor antagonist compound (e.g, CPI-444) at 10 mg, 25 mg or 100 mg, as a
dry powder
mixture of adenosine-A2A (A2A) receptor antagonist compound (e.g. CPI-444)
resinate with
common excipients and packaged in high density polyethylene (HDPE) bottles
fitted with a
polypropylene tamper evident child-resistant cap with an integrated desiccant.
The adenosine-
A2A (A2A) receptor antagonist (e.g, CPI-444) resinate is a complex of the
adenosine-A2A
(A2A) receptor antagonist and a cation exchange resin (Amberlite IRP69Tm). The
ingredients
are listed in Table A.
[0387] Table A: CPI-444 Capsules Table of Ingredients
Ingredient Function
CPI-444 (adenosine-A2A (A2A) receptor antagonist) Active ingredient
Sodium polystyrene stdfonate (Amberlite IRP69) resin Ion-exchange resin,
release modifying agent
Mannitol, spray-dried Diluent
Croscarmellose sodium Disintegrant
Colloidal silicon dioxide Glidant
Sodium stearylfumarate Lubricant
Size 0-elongated HPMC capsule Capsule shell:
mg ¨ opaque, Swedish orange
25 mg ¨ opaque white
100 mg ¨ opaque, Swedish orange
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DETECTING ADENOSINE RECEPTOR ACTIVATION
[0388] Cyclic AMP (cAMP) response element binding protein (CREB) is a cellular

transcription factor. CREB is activated by signaling cascades resultant from
an array of
extracellular signals. One such activating signal cascade is triggered by
agonist binding to
adenosine receptor (e.g. A2A and A2B receptors). Agonist activation of
adenosine receptor
results in activation of CREB by phosphorylation. Agonist activation of
adenosine receptor also
results in activation of protein kinase A (PKA) upstream of CREB.
[0389] Cancer therapies described above which include adenosine receptor
antagonists, alone
or in combination, alter signaling cascades that result in CREB activation
(see FIG. 24).
Detection of downstream effects of treatment with adenosine receptor
antagonists can be assayed
to determine cellular response to treatment. Activation of CREB activation can
be detected by
detection of phosphorylated CREB.
[0390] In embodiments, pCREB is detected by a pCREB detection agent. In
embodiments,
pCREB is detected by an antibody (e.g. a commercially available antibody). In
embodiments, a
pCREB detection agent is detected by Fluorescence-activated cell sorting
(FACS). In
embodiments, pCREB is detected in a subpopulation of cells (e.g. T cells
and/or B cells). In
embodiments, pCREB is detected using an antibody in an ELISA format. In
embodiments,
ELISA detection can be from bulk cell lysate or sorted B cells and/or T cells.
[0391] In embodiments, cells for detection of pCREB can be harvested from
blood (e.g. from
circulating blood). In embodiments, cells for detection of pCREB can be
harvested from a tumor
site. In embodiments, cells for the detection of pCREB are isolated, stained
and fixed. In
embodiments, cell staining is with antibodies against pCREB, CD3, CD4, CD8,
CD27, CD20,
CD45RA, cPARP. In embodiments, cells for the detection of pCREB are sorted by
FACs. In
embodiments, FACS detection of an antibody against CD19 and an antibody
against CD20
indicate a B cell. In embodiment, FACS detection of antibodies against CD3,
CD4 and CD8
indicates a T cell. In embodiments, FACS detection of antibodies against cPARP
indicates an
apoptotic cell. In embodiments, detection of pCREB induction is from an
isolated cell population
(e.g. B cells or T cells).
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[0392] In embodiments, detection of activated PKA is used in addition to, or
as a proxy for,
detection of pCREB.
[0393] CREB activation can be induced by activation of adenosine receptor
agonists including
adenosine, NECA, or analogs thereof NECA is a synthetic adenosine analog. In
embodiments,
NECA is administered to cells in a concentration of about 0.1 M, 0.2 M, 0.3
M, 0.4 M, 0.5
M, 1.0 M, 2 M, 3 M, 4 M, 5 MM, 6 M, 10 M, 15 M, 20 M, 30 M or more to
activate adenosine receptors.
[0394] In embodiments, inhibition of pCREB induction by an adenosine receptor
agonist can
be used as an in vitro screening or evaluation assay to identify and
characterize adenosine
receptor antagonists.
PATIENT SELECTION AND DOSAGE ADJUSTMENT
[0395] In patients treated with adenosine receptor antagonists, alone or in
combination, effects
on downstream effectors (e.g. CREB) can be used to determine treatment or
dosage efficacy of
the adenosine receptor antagonist therapy. Furthermore, assessment of CREB
activation can be
used to determine the diurnal timing of therapy administration.
[0396] An individual patient reaction to treatment with an adenosine receptor
antagonist (e.g.
an A2A receptor or A2B receptor antagonist) can be detected by measuring of
cellular effects.
In embodiments, cellular effects of treatment can be monitored in a patient
sample (e.g. a blood
or tumor sample). In embodiments, a blood sample is used to assay CREB
activation. As
described above adenosine receptor agonists result in CREB activation,
conversely adenosine
receptor antagonists can inhibit the activation of CREB. In embodiments,
monitoring inhibition
of activation of CREB via the adenosine receptor pathway can indicate efficacy
of an adenosine
receptor antagonist.
[0397] In embodiments, cells are isolated from a patient sample (e.g. a blood
or tumor sample).
In embodiments, CREB activation following treatment with an adenosine receptor
agonist (e.g.
NECA) is monitored prior to treatment with an adenosine receptor antagonist
(e.g. detection of
induction of pCREB prior to treatment with CPI-444). In embodiments, pCREB
induction by an
adenosine receptor agonist is assayed relative to a control sample (e.g. cells
treated with PMA).
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In embodiments, an additional sample is collected following treatment with an
adenosine
receptor antagonist, alone or in combination (e.g. CPI-444, or CPI-444
combination therapy with
azetolizumab). In embodiments, a sample is collected after about 7 days, 8
days, 9 days, 10
days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 12 weeks
or more
following treatment with an adenosine receptor antagonist. A comparison can be
made between
pCREB induction by an adenosine receptor agonist prior to and following
treatment with an
adenosine receptor antagonist to determine the degree to which treatment has
reduced
downstream effects of adenosine receptor activation (e.g. pCREB induction).
[0398] In embodiments, subjects who display attenuated induction of pCREB by
an adenosine
receptor agonist following treatment with an adenosine receptor antagonist are
selected as
responsive to treatment with adenosine receptor antagonists. In embodiments,
where attenuation
or inhibition of pCREB induction by NECA by an adenosine receptor antagonist
is incomplete a
dosage of the adenosine receptor antagonist can be increased. In embodiments,
a patient sample
is taken prior to adenosine pathway blockade (e.g. with an adenosine receptor
antagonist) and
treated with an adenosine receptor agonist to detelmine the level of induced
pCREB signaling
which may guide patient selection for treatment by adenosine pathway blockade
(e.g. with an
adenosine receptor antagonist).
[0399] Furthermore, diurnal variations in concentration of an adenosine
receptor antagonist in
the circulation result from one or twice daily administration. This variation
in concentration
throughout the course of the day can impact treatment efficacy. Using methods
and
compositions of the present invention variations of the efficacy of treatment
can be monitored by
detecting pCREB induction by an adenosine receptor agonist at different time
points following
administration of an adenosine receptor antagonist. In embodiments, pCREB
induction by an
adenosine receptor agonist can be monitored at about 0 hours, 0.5 hours, 1
hours, 2 hours 3
hours, 4 hours, 5 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16
hours, 20 hours, or 24
hours following administration of an adenosine receptor antagonist. In
embodiments, timing of
administration of an adenosine receptor antagonist can be altered for maximal
inhibition of
pCREB induction by an adenosine receptor agonist.
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EXAMPLES
EXAMPLE 1
[0400] Binding affinity for human adenosine receptors (V81444-07-076)
[0401] The ability of CPI-444 to displace radioligand binding for the four
identified adenosine
receptor subtypes (Al, A2A, A2B, and A3) was tested in human recombinant
receptors
expressed in mammalian cell lines. Results are shown in Table 1.
[0402] Table 1 Displacement of radioligand binding by CPI-444
Receptor pKi Mean Ki nM Selectivity against
A2A
Adenosine Al 6.72 192 x54
Adenosine A2A 8.45 3.54
Adenosine A2B 5.82 1,528 x431
Adenosine A3 5.61 2,455 x 693
CPI-444 bound A2A receptors with an affinity (Ki) value of 3.54 nM (the
negative logarithm of
Ki [pKi] = 8.45). CPI-444 showed greater than 50 fold selectivity for the A2A
receptor over
other adenosine receptor subtypes.
[0403] Functional activity on human adenosine receptors (V81444-07-078)
[0404] CPI-444 was evaluated in experimental paradigms designed to quantify
antagonist
interactions with the four identified human adenosine receptor subtypes
expressed in Chinese
hamster ovary (CHO-K1) cells. At all concentrations tested, CPI-444 caused a
right-shift in the
agonist concentration-response curve without decreasing the maximum agonist
response,
indicating a competitive mode of action. Antagonist pA2 (negative logarithm of
the antagonist
concentration causing a 2-fold shift in the agonist concentration response
curve [equivalent to 50
% occupancy]) values were estimated from the extent of this right¨shift and
showed V81444 to
be a potent A2A receptor antagonist with a pA2 value of 8.49 (3.2 nM) at the
A2A receptor
(Table 2).
[0405] Table 2 CPI-444 adenosine receptor antagonist activity.
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Receptor pA2 mean pA2 expressed as chemical Selectivity
against
concentration nM A2A
Adenosine Al 6.53 295 x 92
Adenosine A2A 8.49 3.2
Adenosine A2B 6.36 436 x136
Adenosine A3 5.65 2,240 x 700
CPI-444 was more than 90-fold selective for the A2A receptor relative to the
other adenosine
receptors.
[0406] Effect of CPI-444 on cAMP Production (CPI-RSR-003)
Adenosine signaling through A2AR leads to increases in the levels of cAMP.
This study
evaluated the ability of CPI-444 to prevent cAMP production in primary human T
cells
stimulated with NECA, a stable analog of adenosine (CPI-RSR-003).
[0407] T cells were isolated from human PBMC by negative selection and
activated via
CD3/CD28 stimulation for 48 hours to induce A2AR expression. Stimulated T
cells were then
"rested" for 24 hours by removal of CD3/CD28 stimulation in order to minimize
background
levels of cAMP. Rested T cells were incubated in the presence of NECA and CPI-
444 or vehicle
control for 10 minutes prior to measurement of cAMP using the LANCE Ultra cAMP
FRET-
based assay (Perkin Elmer). CPI-444 completely blocked the production of cAMP
upon NECA
treatment at all levels of NECA tested (10-5 to 10-9 M). CPI-444 also
prevented cAMP
production upon NECA stimulation in a dose-dependent manner (FIG. 6). These
results confirm
that CPI-444 is an A2AR antagonist capable of inhibiting cAMP induced by
adenosine signaling.
[0408] Effect of CPI-444 on IL-2 and IFNy secretion (CPI-RSR-002)
104091 The objective of this study was to determine if CPI-444 abrogates the
immunosuppressive effects of adenosine on T cell activation and Thl cytokine
release in vitro
(CPI-RSR-002). Primary human PBMCs were cultured for 1 hour in the presence of
an A2AR
agonist (NECA or CGS21680, 1 04) to simulate the effects of adenosine on
immune cell
function. Purified anti-CD3 and anti-CD28 monoclonal antibodies (1 ug/ml) were
then added to
activate T cells for 48 hours. In this study, AlphaLISA assays (PerkinElmer)
analyzed on an
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EnVision MultiLabel Reader were used to measure cytokine release according to
the
manufacturer's instructions. NECA and CGS21680 suppressed release of the Thl
cytokines IL-2
and IFNy, mimicking the immunosuppressive effects of adenosine signaling (FIG.
7).
[0410] Blockade of A2AR with CPI-444 (1 p.M) prior to T cell activation
neutralized the
immunosuppressive effects of NECA and CGS21680 and restored IL-2 and IFNy
secretion back
to levels observed in the absence of exogenous adenosine signaling (DMSO
control). These
results show that restoration of T cell function is an important mechanism by
which CPI-444
enables an anti-tumor response in vivo.
[0411] CPI-444 Does Not Inhibit Tumor Cell Proliferation In Vitro (CPI444-RSR-
006)
[0412] CPI-444 inhibits the growth of MC38, CT26, and EL4 tumors at either
primary (MC38,
CT26) or metastatic (EL4) sites in syngeneic mouse tumor models. This study
evaluated the
effects of CPI-444 on mouse tumor cell proliferation and viability. MC38,
CT26, and EL4 cells
were cultured in the presence of CPI-444 at a concentrations ranging from 10
CM to 1 pM for 24
hours. Staurosporine, a well-characterized inducer of apoptosis, was included
as a positive
control for cell death. Cell viability/proliferation was measured by XTT. In
this assay, XTT salts
are cleaved by metabolically active (viable) cells, thereby producing a
colorimetric change in the
culture media that can be quantified by measuring absorbance at 405nm and 620
nm on a
spectrophotometer. No significant decrease in the Specific Absorbance
(A450Test - A450Blank -
A620Test) was observed in MC38, CT26, or EL4 cultures at any concentration of
CPI-444 tested
(representative results, FIG. 8). These results indicate that CPI-444 efficacy
observed in vivo is
likely not due to a direct effect on tumor cell proliferation.
[0413] Effect of CPI-444 on pERK levels in human CD4+ cells (CPI-RSR-008)
[0414] This study shows that stimulation of A2AR with an adenosine analog
(NECA) dampens
ERK activation in human PBMCs following TCR cross-linking (CPI-RSR-008). Both
CPI-444
and the A2AR specific antagonist ZM 241385 restore ERK signaling in the
presence of NECA.
The percentage of CD4+ T cells showing TCR-mediated ERK phosphorylation was
reduced in
the presence of NECA (1p.M). Addition of CPI-444 restored pERK levels in a
dose-dependent
manner (FIG. 9). This finding supports a role for CPI-444 in restoring T cell
activation in the
presence of otherwise immunosuppressive levels of adenosine.
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[0415] Effect of CPI-444 on phosphorylation of cAMP response element binding
protein
(pCREB; CPI-RSR-007)
[0416] Adenosine signaling via A2AR leads to an increase in intracellular cAMP
and
subsequent phosphorylation of CREB. This study demonstrates that the adenosine
analog NECA
activates phosho-CREB in fresh PBMCs, primarily in the B cell population (CPI-
RSR-007).
Furthermore, this phosphorylation event is completely inhibited by CPI-444, as
well as by the
known A2AR antagonist ZM 241385 (FIG. 10). This finding demonstrates that CPI-
444 inhibits
NECA-mediated cell signaling through A2AR and provides a functional assay for
CPI-444
activity.
[0417] In Vivo Studies
[0418] Oral administration of CPI-444 at 100 mg/kg or 10 mg/kg significantly
inhibits the
growth of MC38 colon tumors compared to the vehicle control in syngeneic
hosts.
[0419] Oral administration of CPI-444 at 10, 30 or 100 mg/kg produced a
therapeutic response
on established primary tumors in the EL4 syngeneic mouse lymphoma model. A
significant
dose-dependent inhibition of tumor growth within regional lymph nodes was
observed in mice
treated with CPI-444.
[0420] CPI-444 (100 mg/kg) or anti-PD-1 antibody monotherapy inhibits the
growth of CT26
colon tumors in syngeneic hosts. CPI-444 + anti-PD-1 combination therapy
eliminated CT26
tumors in nearly all mice. Combination therapy also produced a significant
increase in long-term
survival compared to either agent administered alone.
[0421] Syngeneic EL4 Mouse Lymphoma Model (CPI-RSR-001)
[0422] This study evaluated the anti-tumor effect of CPI-444 on tumor growth
and metastasis
in a transplanted CD4+ mouse T cell lymphoma model (CPI-RSR-001). Syngeneic
C57BL/6
female mice (8 ¨ 10 weeks old) were injected (via subcutaneous route) with EL4
cells. Tumor-
bearing mice were administered control vehicle (40% Hydroxypropyl Beta-
Cyclodextrin) or
CPI-444 solution daily by oral gavage upon formation of measureable tumors
(140 55 mm3).
CPI-444 doses of 10, 30, and 100 mg/kg were evaluated. CPI-444 treatment
produced a minimal
therapeutic response on established primary tumors. A dose response was
observed, yet all dose
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levels failed to produce a significant inhibition of tumor growth. In
contrast, a significant, dose-
dependent decrease in the number and size of enlarged regional lymph nodes was
observed (FIG.
11), indicating that CPI-444 inhibited or eliminated tumor metastases in this
model.
[0423] Syngeneic MC38 Mouse Colon Carcinoma Model (CPI-RSR-004)
[0424] The objective of this study was to evaluate the anti-tumor activity of
CPI-444 in a
mouse colon carcinoma model (CPI-RSR-004). MC38 colon cancer cells were
subcutaneously
injected onto the backs of syngeneic C57BL/6 mice. One day after tumor cell
engraftment,
vehicle control (40% Hydroxypropyl Beta-Cyclodextrin) or CPI-444 was
administered daily via
oral gavage for 28 days. Administration of CPI-444 at 1 mg/kg did not inhibit
tumor growth,
however doses of 10 mg/kg and 100 mg/kg resulted in a significant inhibition
of tumor growth
(FIG. 12). Notably, complete tumor regression was observed in a subset of mice
within all
cohorts treated with CPI-444 (FIG. 12). It is possible that full tumor
eradication could be
achieved in additional mice with longer administration of CPI-444. These
results demonstrate
that MC38 is responsive to CPI-444 treatment. Syngeneic CT26 Mouse Colon
Cancer Model
with CPI-444 in Combination with anti-PD1 (CPI-RSR-005)
[0425] The objective of this study was to evaluate the effects of CPI-444 in a
transplanted
mouse colon cancer model in combination with a blocking anti-PD-1 monoclonal
antibody (CPI-
RSR-005). CT26 mouse colon cancer cells were engrafted onto the back of
syngeneic male
Balb/c mice. Oral administration of control vehicle (40% solution of
hydroxypropyl-beta-
cyclodextrin) or CPI-444 (100 mg/kg) was initiated the same day tumors were
engrafted (Day 0).
Treatment continued for 12 days. Half of the mice in the vehicle control group
as well as half the
mice in the CPI-444 treatment group received anti-PD-1 mAb (RMP1-14, 100
ug/mouse, i.p.) on
days 7,9, 11, and 13. Administration of anti-PD-1 or CPI-444 resulted in an
inhibition of tumor
growth, however tumors were not completely eradicated by either treatment
(FIG. 13).
Administration of CPI-444 in combination with anti-PD-1 stabilized or
eliminated tumors in 8/9
mice, resulting in improved overall survival for more than 3 weeks following
the last dose of
CPI-444 or anti-PD-1 antibody (FIG. 13).
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EXAMPLE 2
[0426] Biomarker and Clinical Activity of CPI-444, a Novel Small Molecule
Inhibitor of A2A
Receptor (A2AR), in a Phlb Study in Advanced Cancers
[0427] Adenosine is immunosuppressive and is produced at high concentrations
in tumors by
both CD73 and direct release from tumor cells. Adenosine activates A2AR, an
immune
checkpoint that leads to direct suppression of effector T cells and
stimulation of regulatory T
cells. CPI-444 is an oral, selective A2AR inhibitor that has been well
tolerated in Phase (Ph) 1
and 2 studies in non-oncology indications. CPI-444 shows activity in multiple
preclinical tumor
models as a single agent and synergistic efficacy when given in combination
with other
checkpoint inhibitors, including anti-PD-Li.
[0428] CPI-444, with or without the investigational agent atezolizumab (anti-
PD-L1), is being
studied in an ongoing Phlb trial in solid tumor patients (pts). Pts with
either lung, melanoma,
triple negative breast, bladder, colorectal, renal, or head and neck cancers
are treated at various
doses of either single agent CPI-444 or combined with atezolizumab. After a
dose selection
stage, pts are treated in 10 disease specific cohorts (5 single agent and 5
combination). Cohorts
may be expanded based on response criteria: complete response, partial
response or stable
disease (SD). Biomarkers are evaluated including immune cells by flow
cytometry in peripheral
blood and pre/post treatment tumor biopsies as well as adenosine pathway
modulation by
immunohistochemistry and gene expression.
[0429] In 7 pts treated to date, CPI-444 has been well tolerated with no Grade
3 or 4 treatment
related adverse events. 2 pts (1 combination and 1 single agent) have reached
the first efficacy
assessment by CT and both demonstrated SD (unconfirmed at 2 months); these 2
pts, and 4
others who have not yet reached efficacy evaluation, remain on treatment.
[0430] In the two pts with SD, peripheral blood showed increases in PD-1+CD8+
cells (1.7
and 2.4 fold compared to baseline). This is consistent with preclinical models
and reflects
effector T cell activation, similar to reports by others in patients treated
with anti-PD-Li.
[0431] CPI-444 is well tolerated and demonstrates biological activity
indicating activation of T
cell immunity. This is the first demonstration of treatment-associated immune
modulation in
cancer patients receiving an adenosine antagonist.
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EXAMPLE 3
[0432] Adenosine A2A receptor antagonist, CPI-444, blocks adenosine-aediated T
cell
suppression and exhibits anti-tumor activity alone and in combination with
anti-PD-1 and anti-
PD-Li
[0433] Elevated extracellular adenosine in the tumor microenvironment
generates an
immunosuppressive niche that promotes tumor growth and metastasis. Adenosine
signaling via
A2A receptor (A2AR) on immune cells suppresses anti-tumor immunity and may
also limit
efficacy of immunotherapies such as anti-PD-Li and anti-PD-1 antibodies.
[0434] CPI-444 is a potent, oral, selective A2AR antagonist that has been well
tolerated in Ph
1 and 2 studies in non-oncology indications. Efficacy of CPI-444 was evaluated
in MC38 and
CT26 syngeneic mouse tumor models. hi MC38, daily treatment of mice with CPI-
444 (1, 10,
100 mg/kg) led to dose-dependent inhibition of tumor growth, leading to tumor
elimination in
9/30 mice. Combining CPI-444 with anti-PD-Li treatment in MC38 synergistically
inhibited
tumor growth and eliminated tumors in 90% of treated mice. In an additional
model, CT26, CPI-
444 alone or anti-PD-1 alone led to non-significant reductions in tumor
growth; however, the
combination of CPI-444 and anti-PD-1 led to a synergistic inhibition of tumor
growth and
prolonged survival compared to either agent alone.
[0435] When cured mice were later re-challenged with MC38 cells, tumor growth
was fully
inhibited, indicating that CPI-444 induced systemic anti-tumor immune memory.
CD8+ T cell
depletion abrogated the efficacy of CPI-444 anti-PD-Li treatment,
demonstrating a role for
CD8+ T cells in mediating primary and secondary immune responses.
[0436] Anti-tumor efficacy of CPI-444 anti-PD-Li was associated with
increased CD8+ cell
infiltration and activation in MC38 tumor tissues. Additionally, levels of
immune checkpoints
were modulated by treatment with CPI-444, including GITR, 0X40, and LAG3 on
tumor
infiltrating lymphocytes and circulating T cells, suggesting a broad role for
adenosine mediated
immunosuppression.
[0437] Based on these results and others, Applicants have initiated a Phase lb
clinical trial to
examine safety, tolerability, biomarkers, and preliminary efficacy of CPI-444
as a single agent
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and in combination the investigational anti-PD-Li antibody, Atezolizumab, in
patients with solid
tumors.
EXAMPLE 4
[0438] CPI-444: A Potent and Selective Inhibitor of Adenosine 2A Receptor
(A2AR) Induces
Anti-Tumor Responses Alone and in Combination with Anti-PD-Li.
[0439] Adenosine is immune-suppressive, acting through adenosine 2A receptor
(A2AR)
which is expressed on cytotoxic, helper and regulatory T cells, as well as NK,
dendritic and
myeloid derive suppressor cells. CPI-444 is an oral, selective inhibitor of
A2AR that is active as
a single agent in multiple syngeneic mouse models and is synergistic when
combined with anti-
PD-1 or anti-PD-Li antibodies in these models. 75 subjects were previously
dosed with CPI-444
in non-oncology trials, and CPI-444 well was well tolerated with no
significant adverse events
noted. A Phase 1/1b study was initiated that explores safety and efficacy of
CPI-444 as asingle
agent as well as in combination with the anti-PD-L1 antibody TECENTRIQ
(atezolizumab) in
selected histologies.
[0440] In step 1 of the trial, patients were dosed with either 100mg BID for
14 days out of a 28
day cycle, 100mg BID for 28 days, 200mg QD for 14 days or 50mg or 100mg BID
for 14 days in
combination with TECENTRIQ (840mg Q2W). Pharmacodynamic analysis was
conducted on
peripheral blood cells to inform dose selection. Step 1 of Phaselilb was fully
enrolled (n=48)
and dose was selected (100mg BID) based on pharmacodynamic analysis of A2AR
pathway.
Complete inhibition observed at 100mg BID dose that is sustained in 28 day
continuous dosing
cohort. Increases in activated CD8 cell frequencies were observed in patients
treated with single
agent CPI-444 and combined with TECENTRIQ , suggesting immune activation in
response to
treatment (FIG. 19A). TCR repertoire changes were induced in peripheral blood
by single agent
CPI-444 in patient subsets, including patients refractory to prior anti-PD-1
therapy (FIG.s 20A-
20C).
[0441] High TCR diversity (low clonality) at baseline and changes in TCR
repertoire
following treatment show association with early efficacy data in anti-PD-1
naive and refractory
patients. A similar rate of stable disease was observed in anti-PD-1
refractory patients and in
both PD-Li positive and negative patient subsets (FIG.s 21A and 21B). This is
the first
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demonstration of immune cell activation and anti-tumor activity in patients
receiving an
adenosine antagonist.
EXAMPLE 5
[0442] MC38 mouse colon cancer cells were engrafted onto the back of syngeneic
C57BL/6
mice. Oral administration of control vehicle or CPI-444 (100 mg/kg) was
initiated 9 days after
tumors were engrafted (Day 0). Treatment continued for 12 days. Half of the
mice in the
vehicle control group as well as half the mice in the CPI-444 treatment group
received anti-PD-
Li mAb (10F.9G2, 200 ug/mouse, i.p.) on days 9, 12, 15, and 18. 100 ug of Anti-
mCD4 (Clone
GK1.5) was administered on days 8, 11, 14, and 17, and 500 ug of Anti-mCD8
(Clone 53-6.72)
was administered on days 8 and 15. T cell depletion was verified by flow
analysis. FIG.s 23A
and 23B show tumor volume at different time points since engraftment for the
dosing cohorts.
These results suggest CD8+ T cells are required for the efficacy of CPI-444
alone or in
combination with Anti-PD-Li.
EXAMPLE 6
[0443] MC38 mouse colon cancer cells were engrafted onto the back of syngeneic
C57BL/6
mice. Oral administration of control vehicle or CPI-444 (100 mg/kg) was
initiated 7 days after
tumors were engrafted (Day 0) (FIG. 23C). Treatment continued for more than 9
days. Half of
the mice in the vehicle control group as well as half the mice in the CPI-444
treatment group
received anti-PD-Li mAb (10F.9G2, 200 ug/mouse, i.p.) on days 7, 10, 13, and
16. 100 ug of
Anti-mCD4 (Clone GK1.5) and/or 500 ug of Anti-mCD8 (Clone 53-6.72) was
administered on
day 6. T cell depletion was verified by flow analysis. FIG.s 23A and 23B show
tumor volume at
different time points since engraftment for the dosing cohorts. These results
suggest CD8+ T
cells are required for the efficacy of CPI-444 alone or in combination with
Anti-PD-Li.
EXAMPLE 7
[0444] Materials and Methods
Whole Blood Processing
[0445] Blood samples were derived from patients and processed using the
following protocol:
= Whole blood in heparin delivered overnight and assay begins the next
morning
= Aliquot 67.5 uL blood per well and recover at 37C for lhr
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= Add 7.5 uL of NECA or PMA per well for 15 minutes at 37C
o NECA at 1, 3 or 10 uM
= Fix cells with 1.5mL of BD Lyse/Fix buffer according to manufacturer
= Spin and resuspend in lmL cold Me0H and store -80C.
Antibody Staining
[0446] Fix cells derived from patient blood were stained using the following
protocol:
= Spin out of Me0H
= Wash 2X with FACS buffer (phosphate buffered saline containing 1% bovine
serum
albumin and 0.1% sodium azide)
= Stain 1 hour
o Antibody cocktail:
= pCREB Alexa Flour647 (Cell Signaling Technology Cat. No. 14001S)
= CD3 Horizon V500 (BD Cat. No. 561416)
= CD4 Brilliant Violet 421 (BD Cat. No. 562424)
= CD8 PerCP-Cy5.5 (BD Cat. No. 560662)
= CD27 FITC (BD Cat. No. 340424)
= CD20 PE (BD Cat. No. 561174)
= CD45RA PE-Cy7 (BD Cat. No. 649457)
= cPARP Alexa Flour700 (BD Cat, No, 560640)
= Wash 2X with FACS buffer
= Fix cells with 1.6% paraformaldehyde (PFA) for 5 minutes at ambient
temperature
= Spin, aspirate, and bring cells to acquisition volume in 1.6% PFA
= Acquire cells on flow cytometer.
EXAMPLE 8
[0447] Induction of pCREB by NECA in B cells harvested from whole blood and
stained as
described above was monitored. Two subjects were assayed, one treated with
200QD CPI-444
(FIG. 27) and a subject treated with 50BID CPI-444 + Atezolizumab (atezo)
(FIG. 28) prior to
treatment and at 2 time points after 14 days of treatment. NECA was used to
stimulate CREB
activation via the adenosine receptor pathway in concentrations of luM NECA,
(sub-saturation,
where inhibition is expected), and 10uM NECA (saturation, where NECA may out-
compete CPI-
444 resulting in inhibition). Control samples were treated with phorbol
myristate acetate (PMA).
[0448] Baseline induction of pCREB following NECA treatment is shown at C1D1
prior to
treatment. NECA induction of pCREB was then monitored at C1D14 at Ohr (trough)
prior to
CPI-444 or CPI-444 + atezo treatment, when CPI-444 concentration in
circulating blood is at its
lowest (see FIG. 26). The second time point is C1D14, 1.5 hours after
administration of CPI-444
or CPI-444 + atezo treatment.
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[0449] In FIG. 27, partial inhibition of the adenosine receptor activation by
CPI-444 is seen by
the inhibition of the NECA induced increase of pCREB in B cells treated with 1
p.M NECA. In
Fig. 28, near complete inhibition of adenosine receptor activation by CPI-444
+ atezo is seen by
the inhibition of the NECA induced increase of pCREB in B cells treated with
ItiM NECA.
EXAMPLE 9
[0450] Induction of pCREB by NECA in B cells (FIG. 29 and FIG. 30) and T cells
(FIG. 31)
harvested from whole blood and stained as described above was monitored. Two
subjects were
assayed, one treated with 200QD CPI-444 and a subject treated with 50BID CPI-
444 +
Atezolizumab (atezo) prior to treatment and at multiple time points after 14
days of treatment.
NECA was used to stimulate CREB activation via the adenosine receptor pathway
in
concentrations of luM, 3uM, and 10uM. Unstimulated control samples were
treated with
phorbol myri state acetate (PMA).
[0451] pCREB induction was measured at day 1 pre-treatment and a day 14 of
treatment at
trough (prior to treatment administration), 1.5hours, 3 hours, 5 hours, and 8
hours following
administration of CPI-444 or CPI-444 +atezo.
[0452] pCREB induction by NECA is attenuated following 14 days of treatment of
CPI-444
and CPI-444 + atezo. This attenuation is clearest with treatment of sub-
saturation NECA (luM,
and 3uM) in B cells. The 14 day time points show the maximal inhibition at
1.5hr ¨ 3hr and the
minimal inhibition at trough and thus reveal the degree of inhibition across
time (e.g. whether
maximal inhibition is maintained over time).
EXAMPLE 10
[0453] Biomarkers were analyzed in archival tumor tissue and serial biopsies
as well as in
peripheral blood to determine whether CPI-444 affects peripheral and intra-
tumoral immune
activation and T cell repertoires and to identify markers that are associated
with efficacy.
EXAMPLE 11
[0454] On day 1, prior to drug therapy, blood was collected and stimulated
with NECA to
induce CREB phosphorylation (pCREB) (FIG.s 24 and 25) and the level of
uninhibited signaling
in B cells and T cells was deteltnined (FIG.s 26-28). On day 14, blood was
collected prior to
dosing (Ohr, trough) and for a time course post-dose (1.5,3, 5.5, 8hr) (FIG.s
26-28). The level of
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signaling was determined and the percent inhibition on Day 14 compared to Day
1 pre-treatment
was calculated.
[0455] The relative amount of pCREB inhibition was assessed for each dosing
cohort. The
majority of patients in the 100mg BID cohort had the highest pCREB inhibition
at trough and
near complete inhibition after taking their morning dose (FIG.s 34B). Little
fluctuation from
trough to peak in the 100mg BID dosing group was observed, demonstrating that
peak inhibition
(at the 3hr time of peak plasma drug levels) is maintained through drug trough
and therefore
100mg BID is an appropriate dose for continuous functional inhibition (FIG.
34D). The 50mg
BID is not high enough for sustained inhibition (FIG. 34A), and the 200mg QD
dose achieves
high peak levels but is not maintained at trough as CPI-444 is administered
only once per day
(FIG. 34C).
[0456] Assessing pharmacokinetics and pharmacodynamics revealed a relationship
between
pCREB percent inhibition and plasma levels of CPI-444. For CPI-444 plasma
levels greater than
2,000 ng/mL, near complete inhibition of pCREB was observed in both B cells
(FIG. 35A) and T
cells (FIG. 35B).
[0457] Applicants observed a strong correlation between inhibition of pCREB in
B cells and
inhibition of pCREB in CD4+ T cells (FIG. 36). In this assay, the pCREB signal
is stronger in B
cells than CD4+ T cells, thus the signal-to-noise is better in B cells. This
shows that at the
population level, pCREB inhibition in B cells is an appropriate surrogate for
directly measuring
pCREB in T cells, a cell type of interest for CPI-444 activity.
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EMBODIMENTS I
[0458] Embodiment 1. A method of treating cancer in a subject in need
thereof, said
method comprising administering to said subject a therapeutically effective
amount of an
adenosine-A2A (A2A) receptor antagonist and a programmed cell death protein 1
(PD-1)
signaling pathway inhibitor.
104591 Embodiment 2. The method of embodiment 1, wherein said A2A receptor
antagonist is a compound of formula:
RI
N
Na
N
R3 (1),
wherein,
R1 is independently hydrogen, halogen, -CV3, -CN, -S02C1, -SO0R9,
-SO,INR9R NHC=(0)NHNH2, -NHC=(0)NR9Rto,
-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9R1o, -0R9, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl;
R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -S0,12R11,
-S0,2NRIIR12, _NHNH2, _mic=(0)NHNH2,
-NHC=(0)NR IR12, _N(0)m2, NR" R'2, _coy-K _
C(0)-0R11, -C (0)NR11R12,
-0R11, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R3 is independently hydrogen, halogen, -CX'3, -CN, -502C1, -S0,13R13,
-S0,3NR13R14, _NHNH2, _0NRI3R14, _
NHC-(0)NHNH2, -NHC-(0)NR13R14,
_N(0)m3,

_NR13R14, _m_t_o_R13, _copy-13, _
C(0)-0R13, -C(0)NR13R14, _0-IC13, = ,
substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
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cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl;
R9, RH), Rn, R12, R13 and R'4
are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NIIC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl;
Xb and X' are independently -F, -Cl, -Br, or -I;
ni, n2 and n3 are independently an integer from 0 to 4;
ml, m2 and m3 are independently an integer from 1 to 2; and
Vi, v2 and v3 are independently an integer from 1 to 2.
[0460] Embodiment 3. The method of embodiment 2, wherein said A2A receptor
antagonist is a compound of formula:
R1
N/
N
NH2
R5 2
0 -R6
R5 (II),
wherein
R6, R61 and R62 are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NI-I2, -NHNH2, -ONH2, -NTIC=(0)NHNH2, substituted or unsubstituted
alkyl, substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
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[0461] Embodiment 4. The method of one of embodiments 1-3, wherein said A2A

receptor antagonist is a compound of formula:
N,
IN
NH2
-N
0
[0462] Embodiment 5. The method of one of embodiments 1-4, wherein said PD-
1
signaling pathway inhibitor is a programmed death-ligand 1 (PD-L1) antagonist
or a PD-1
antagonist.
[0463] Embodiment 6. The method of embodiment 5, wherein said programmed
death-
ligand 1 (PD-L1) antagonist is an antibody or a small molecule.
[0464] Embodiment 7. The method of embodiment 6, wherein said PD-Li
antagonist is an
antibody.
[0465] Embodiment 8. The method of embodiment 7, wherein said antibody is
atezolizumab.
[0466] Embodiment 9. The method of embodiment 5, wherein said PD-1
antagonist is an
antibody or a small molecule.
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[0467] Embodiment 10. The method of any one of embodiments 1-9, wherein
said A2A
receptor antagonist and said PD-1 signaling pathway inhibitor are administered
in a combined
synergistic amount.
[0468] Embodiment 11. The method of one of embodiments 1-10, wherein said
A2A
receptor antagonist and said PD-1 signaling pathway inhibitor are administered
simultaneously
or sequentially.
[0469] Embodiment 12. The method of one of embodiments 1-11, wherein said
A2A
receptor antagonist is administered at a first time point and said PD-1
signaling pathway inhibitor
is administered at a second time point, wherein said first time point precedes
said second time
point.
[0470] Embodiment 13. The method of embodiment 12, wherein said second time
point is
within less than about 120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14,
13, 12, 10, 11,9, 8, 7, 6,
5, 4, 3, 2 or 1 days from said first time point.
[0471] Embodiment 14. The method of embodiment 12 or13, wherein said second
time
point is within about 8, 10 or 12 days from said first time point.
[0472] Embodiment 15. The method of one of embodiments 1-11, wherein said
PD-1
signaling pathway inhibitor is administered at a first time point and said A2A
receptor antagonist
is administered at a second time point, wherein said first time point precedes
said second time
point.
[0473] Embodiment 16. The method of embodiment 15, wherein said second time
point is
within less than about 120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14,
13, 12, 10, 11, 9, 8, 7, 6,
5, 4, 3, 2 or 1 days from said first time point.
[0474] Embodiment 17. The method of embodiment 15 or16, wherein said second
time
point is within about 8, 10 or 12 days from said first time point.
[0475] Embodiment 18. The method of one of embodiments 1-17, wherein said
A2A
receptor antagonist is administered at an amount of about 0.5 mg/kg, 1 mg/kg,
5 mg/kg, 10
mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg,
90 mg/kg,
100 mg/kg, 200 mg/kg or 300 mg/kg.
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[0476] Embodiment 19. The method of one of embodiments 1-18, wherein said
A2A
receptor antagonist is administered at an amount of about 1 mg/kg.
[0477] Embodiment 20. The method of one of embodiments 1-18, wherein said
PD-1
signaling pathway inhibitor is administered at an amount of less than about
1,300 mg.
[0478] Embodiment 21. The method of one of embodiments 1-20, wherein said
PD-1
signaling pathway inhibitor is administered at an amount of about 1,200 mg.
[0479] Embodiment 22. The method of one of embodiments 1-21, wherein said
cancer is
selected from lung cancer, bladder cancer, melanoma, renal cell carcinoma,
colon cancer,
ovarian cancer, gastric cancer, breast cancer, head and neck carcinoma,
prostate cancer and a
hematologic malignancy.
[0480] Embodiment 23. A method of treating cancer in a subject in need
thereof, said
method comprising administering to said subject a therapeutically effective
amount of an
adenosine-A2A (A2A) receptor antagonist, wherein said A2A receptor antagonist
is a compound
of follnula:
12`
R3 (I),
wherein,
R1 is independently hydrogen, halogen, -CX23, -CN, -S02C1, -SOr1R9,
-S0,,NR9R1 , ¨NHNH2, ¨0NR9Rio, NHC=(0)NHNH2, ¨NHC=(0)NR9Rio, _N(c)rni,_NR9Rio,

-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9R1 -0R9, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl;
R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -S0,12R11,
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-S0v2NRitR12, _NH-m12, _0NR11R12, _NIc=(0)NHNH2,
-NHC=(0)NRIIR12, _N(0).2, _NRI -NH-O-R", _C(0)-0R11, -C(0)NR11R12,
-0R11, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R3 is independently hydrogen, halogen, -CX'3, -CN, -S02C1, -S0n3R13,
-S0,3NR13R14, _NHNH2, _0NRI3R14, _
NHC-(0)NHNH2, -NHC-(0)NR13R14,
-N(0)m3, -
NR13R14, -NTAD-R13, _c(0)-
K C(0)-
0R13, -C(0)NR13R14, _orsIC 13, substituted
or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl;
R9, RI , Rn, R12, -13
K and R14 are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl;
Xb and X' are independently -F, -Cl, -Br, or -I;
ni, n2 and n3 are independently an integer from 0 to 4;
ml, m2 and m3 are independently an integer from 1 to 2; and
vi, v2 and v3 are independently an integer from 1 to 2.
[0481] Embodiment 24. The
method of embodiment 23, wherein said A2A receptor
antagonist is a compound of formula:
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R
/
R6 2
R6
R6
wherein
R6, R61 and R6'2 are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NTC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
[0482] Embodiment 25. The method of embodiment 23 or 24, wherein said A2A
receptor
antagonist is a compound of formula:
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N,
N
\N H2
-N
0
[0483] Embodiment 26. The method of one of embodiments 23-25, further
comprising
administering a therapeutically effective amount of a PD-1 signaling pathway
inhibitor.
[0484] Embodiment 27. The method of embodiment 26, wherein said A2A
receptor
antagonist and said PD-1 signaling pathway inhibitor are administered in a
combined synergistic
amount.
[0485] Embodiment 28. The method of one of embodiments 26-27, wherein said
A2A
receptor antagonist and said PD-1 signaling pathway inhibitor are administered
simultaneously
or sequentially.
[0486] Embodiment 29. The method of one of embodiments 26-28, wherein said
A2A
receptor antagonist is administered at a first time point and said PD-1
signaling pathway inhibitor
is administered at a second time point, wherein said first time point precedes
said second time
point.
[0487] Embodiment 30. The method of embodiment 29, wherein said second time
point is
within less than about 120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14,
13, 12, 10, 11, 9, 8, 7, 6,
5, 4, 3, 2, or 1 days from said first time point.
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[0488] Embodiment 31. The method of embodiment 29 or 30, wherein said
second time
point is within about 8, 10 or 12 days from said first time point.
[0489] Embodiment 32. The method of one of embodiments 26-28, wherein said
PD-1
signaling pathway inhibitor is administered at a first time point and said A2A
receptor antagonist
is administered at a second time point, wherein said first time point precedes
said second time
point.
[0490] Embodiment 33. The method of embodiment 32, wherein said second time
point is
within less than about 120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14,
13, 12, 10, 11, 9, 8, 7, 6,
5, 4, 3, 2, or 1 days from said first time point.
[0491] Embodiment 34. The method of embodiment 32 or 33, wherein said
second time
point is within about 8, 10 or 12 days from said first time point.
[0492] Embodiment 35. The method of one of embodiments 23-34, wherein said
A2A
receptor antagonist is administered at an amount of about 0.5 mg/kg, 1 mg/kg,
5 mg/kg, 10
mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg,
90 mg/kg,
100 mg/kg, 200 mg/kg or 300 mg/kg.
[0493] Embodiment 36. The method of one of embodiments 23-35, wherein said
A2A
receptor antagonist is administered at an amount of about 1 mg/kg.
[0494] Embodiment 37. The method of one of embodiments 23-35, wherein said
PD-1
signaling pathway inhibitor is administered at an amount of less than about
1,300 mg.
[0495] Embodiment 38. The method of one of embodiments 23-37, wherein said
PD-1
signaling pathway inhibitor is administered at an amount of less than about
1,200 mg.
[0496] Embodiment 39. The method of one of embodiments 23-38, wherein said
cancer is
selected from lung cancer, bladder cancer, melanoma, renal cell carcinoma,
colon cancer,
ovarian cancer, gastric cancer, breast cancer, head and neck carcinoma,
prostate cancer and a
hematologic malignancy.
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104971 Embodiment 40. A method of activating a T cell, said method
comprising
contacting said T cell with an A2A receptor antagonist, wherein said A2A
receptor antagonist is
a compound of formula:
R1
R3
wherein,
R1 is independently hydrogen, halogen, -CV3, -CN, -S02C1, -S01R9,
-SOvINR9R1o, _NHNH2, _0NR9Rio, _NHc="NHNH2, _NHc="NR9R1o, _N(0).1,_NR9Rio,
-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9K _0R9 , substituted or unsubstituted
alkyl,
-
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl;
R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -S0,12R11,
-S0v2NR11R12, NHNH2, 0NRIIR12, NHc_(0)NHNH2,
-NHC=(0)NR11Rt2, _N(0).2, _NRI1R12, _NH_o_Rii, K _c(0)- 1,
C(0)-0R11, -C(0)NR11R12,
-OR", substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R3 is independently hydrogen, halogen, -CX`3, -CN, -S02C1, -S0n3R13,
-S0v3NR13R14, _NHNH2, _0NR13R14, _NHc=(0)NHNH2, _NHc=(0)NRI3R14,
-N(0)m3, -
NRI3R14, -C(0)R'3,
C(0)-0R13, -C(0)NR13R14, _oR13, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl;
R9, Rlo, R12, R13 and R'4
are independently hydrogen, halogen, =0, -S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NHC-(0)NHNH2, substituted or unsubstituted alkyl,
substituted
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or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl;
Xb and X' are independently ¨F, -Cl, -Br, or ¨I;
ni, n2 and n3 are independently an integer from 0 to 4;
mt, m2 and m3 are independently an integer from 1 to 2; and
v1, v2 and v3 are independently an integer from 1 to 2.
[0498] Embodiment 41. The method of embodiment 40, further comprising
contacting said
T cell with a PD-1 signaling pathway inhibitor.
[0499] Embodiment 42. The method of embodiment 41, wherein said PD-1
signaling
pathway inhibitor is an antibody or a small molecule.
[0500] Embodiment 43. The method of one of embodiments 40-42, wherein said
T cell is
an effector T cell or a natural killer cell.
[0501] Embodiment 44. The method of one of embodiments 40-43, wherein said
T cell is
an adenosine-suppressed T cell.
[0502] Embodiment 45. The method of one of embodiments 40-44, wherein said
T cell is a
CD8 T cell.
[0503] Embodiment 46. The method of embodiment 45, wherein said CD8 T cell
is a
CD45RA-negative CD8 Tcell.
[0504] Embodiment 47. The method of one of embodiments 40-42, wherein said
T cell is a
CD4 T cell.
[0505] Embodiment 48. The method of embodiment 47, wherein said CD4 T cell
is a
CD45RA-negative CD4 Tcell.
[0506] Embodiment 49. The method of one of embodiments 40-48, wherein said
T cell is
within a subject.
[0507] Embodiment 50. The method of embodiment 49, wherein said subject is
a cancer
subject.
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[0508] Embodiment 51. The method of embodiment 50, wherein said cancer
subject is an
anti-PD-1 refractory subject.
[0509] Embodiment 52. A method of inhibiting A2A receptor activity of a
cell, said method
comprising contacting said cell with an A2A receptor antagonist, wherein said
A2A receptor
antagonist is a compound of formula:
RI
Nah
N R2
(I),
wherein,
RI is independently hydrogen, halogen, -CV3, -CN, -S02C1, -S0mR9,
-SOvINR9Rio, _NHNH2, _coNR9Rto, _NHc="NHNH2, _NHc="NR9Rio, _N(0).1,_NR9Rio,
-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9--K _0R9 , substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl;
R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -S0,12R",
-S0v2NRI1R12, _NHNH2, _0NRIIR12, _NHc=(0)NHNH2,
-NHC=(0)NR tiRt2, _N(0)m2, NR" R'2, _NH_o_Rii, _
C(0)-0R11, -C(0)NRI1R12,
-OR", substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R3 is independently hydrogen, halogen, -CX`3, -CN, -S02C1, -S0n3R13,
-S0,3NR13R14, _NHNH2, _0NRI.3R14, _NHc=(0)NHNH2, _NHc=(0)NRi3R14,
-N(0)m3, -
NRI3R14, _NH_O_R13, _c(or13,
K C(0)-0R13, -C(0)NR13R14, _coR13,
substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl;
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Ro, Rio, R", R12, - 13
K and RIA are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CON-Hz, -OH, -SH, -S02C1, -S0314, -SO4H, -
SO2NF12,
-NO2, -N}I1NE12, -ONE12, -NHC=(0)NHNI-12, substituted or unsubstituted
alkyl, substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl;
X1) and X' are independently ¨F, -Cl, -Br, or ¨I;
n1, nz and n3 are independently an integer from 0 to 4;
mi, mz and m3 are independently an integer from 1 to 2; and
v1, vz and v3 are independently an integer from 1 to 2.
[0510] Embodiment 53. The method of embodiment 52, wherein said A2A receptor
antagonist is a compound of formula:
-N
0
[0511] Embodiment 54. The method of embodiment 52 or 53, wherein said
contacting
comprises binding said A2A receptor antagonist to an A2A receptor of said
cell.
[0512] Embodiment 55. The method of any one of embodiments 52-54, wherein
said cell is
a T cell.
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[0513] Embodiment 56. The method of embodiment 55, wherein said T cell is
an effector T
cell or a natural killer cell.
[0514] Embodiment 57. The method of embodiment 55, wherein said T cell is a
CD8 T
cell.
[0515] Embodiment 58. The method of embodiment 57, wherein said CD8 T cell
is a
CD45RA-negative CD8 Tcell.
[0516] Embodiment 59. The method of embodiment 55, wherein said T cell is a
CD4 Tcell.
[0517] Embodiment 60. The method of embodiment 59, wherein said CD4 T cell
is a
CD45RA-negative CD4 Tcell.
[0518] Embodiment 61. The method of one of embodiments 55-60, wherein said
T cell is
within a subject.
[0519] Embodiment 62. The method of embodiment 61, wherein said subject is
a cancer
subject.
[0520] Embodiment 63. The method of embodiment 62, wherein said cancer
subject is an
anti-PD-1 refractory subject.
[0521] Embodiment 64. A method of increasing an anti-tumor immune response
in a
subject in need thereof, said method comprising administering to said subject
a therapeutically
effective amount of an adenosine-A2A (A2A) receptor antagonist and a
programmed cell death
protein 1 (PD-1) signaling pathway inhibitor.
[0522] Embodiment 65. A method of increasing an anti-tumor immune response
in a
subject in need thereof, said method comprising administering to said subject
a therapeutically
effective amount of an adenosine-A2A (A2A) receptor antagonist, wherein said
A2A receptor
antagonist is a compound of fomiula:
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RI
2
R3 (0,
wherein,
is independently hydrogen, halogen, -CXa3, -CN, -S02C1, -SOõIR9,
-S0,4NR9Rio, _NHNH2, _0NR9Rio, _NHc=.(0)NHNH2, _NHc=,(0)NR9Rio, _
N(0)õ,1,-
NR9Rio,
-C(0)R9, -C(0)-0R9, -C(0 )NR9- Kio, -OR 9, substituted or unsubstituted alkyl,

substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl;
R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -S0n2R11,
-S0,2NRI1R12, _NHNH2, tR12, _
NHC-(0)NHNH2,
-NHC-(0)NR C(0)-OR", -C(0)NR11R12,
-OR", substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R3 is independently hydrogen, halogen, -CX'3, -CN, -S02C1, -S0õ3R13,
-S0,3NR13R14, _NHNH2, _oNRI3R14, _NHc=(0)NHNH2, _NHc=(0)NRI3R14,
-N(0)1,3, -NR13R14, -NH-O-R13, -C(0)R13, -C(0)-0R13, -C(0)NRI3R14, _0- i3, ,
,
suDstitutea or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl;
R9, Rio, RI.% R12, RD and R'4
are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NI-I2, -NHNH2, -ONH2, -NTIC=(0)NtINH2, substituted or unsubstituted
alkyl, substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl;
Xa, Xb and X' are independently -F, -Cl, -Br, or -I;
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n1, n2 and n3 are independently an integer from 0 to 4;
ml, m2 and m3 are independently an integer from 1 to 2; and
Vi, v2 and v3 are independently an integer from 1 to 2.
[0523] Embodiment 66. The method of embodiment 65, wherein said A2A
receptor
antagonist is a compound of formula:
N
N,
\NNNH2
¨N
0
[0524] Embodiment 67. The method of embodiment 65 or 66, further comprising

administering a therapeutically effective amount of a PD-1 signaling pathway
inhibitor.
[0525] Embodiment 68. The method of embodiment 67, wherein said PD-1
signaling
pathway inhibitor is a PD-Li antagonist.
[0526] Embodiment 69. The method of embodiment 68, wherein said PD-Li
antagonist is a
small molecule or an antibody.
[0527] Embodiment 70. A method of increasing the amount of CD8-positive
cells relative
to the amount of regulatory T cells in a subject in need thereof, said method
comprising
administering to said subject a therapeutically effective amount of an
adenosine-A2A (A2A)
receptor antagonist and a programmed cell death protein 1 (PD-1) signaling
pathway inhibitor.
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[0528] Embodiment 71. A method of increasing the amount of CD8-positive
cells relative
to the amount of regulatory T cells in a subject in need thereof, said method
comprising
administering to said subject a therapeutically effective amount of an
adenosine-A2A (A2A)
receptor antagonist, wherein said A2A receptor antagonist is a compound of
formula:
R2
R3 (D,
wherein,
RI- is independently hydrogen, halogen, -CV, -CN, -S02C1, -SOritR9,
-S0,1 NHC-(0)NHNH2, -NHC-(0)N-R9Rio, _N(0).1,_NR9Rio,
-C(0)R9, -C(0)-0R9, -C(0)NR9-K io,
-OR9, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl;
R2 is independently hydrogen, halogen, -CX3, -CN, -S02C1, -S0,12R11,
-S0,2NRiiR12, _NHNH2, _0NR iR12, _NHc_(0)NHNH2,
-NHC=(0)NRi iR12, _N(0).2, NR" R'2, _NH_0_Ri 1, K _c(0)- _
C(0)-0R11, -C(0)NR' 1R12,
-OR", substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R3 is independently hydrogen, halogen, -CX'3, -CN, -S02C1, -S0,13R13,
-S0v3NR NHNH2, 0NR i3R1,1, NHic (0)miNTH2, N-Hc (0)NR13R14,
-NOW, -
NR13R14, _NH-0-R'3,
C(0)R13, -C(0)-0R13, -C(0)NR13IC _ORI3, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl;
R9, Rio, Ri2, - 13
K and R14 are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2N112,
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-NO2, -N112, -NHNI2, -ONH2, -NTC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl;
Xb and X' are independently ¨F, -Cl, -Br, or ¨I;
ni, n2 and n3 are independently an integer from 0 to 4;
ml, m2 and m3 are independently an integer from 1 to 2; and
Vi, v2 and v3 are independently an integer from 1 to 2.
[0529] Embodiment 72. The method of embodiment 71, wherein said A2A
receptor
antagonist is a compound of formula:
_(
N
\\NNtsjui
....2
¨N
0
[0530] Embodiment 73. The method of embodiment 71 or 72, further comprising

administering a therapeutically effective amount of a PD-1 signaling pathway
inhibitor.
[0531] Embodiment 74. The method of embodiment 73, wherein said PD-1
signaling
pathway inhibitor is a PD-Ll antagonist.
[0532] Embodiment 75. The method of embodiment 74, wherein said PD-Li
antagonist is a
small molecule or an antibody.
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[0533] Embodiment 76. A method of decreasing tumor volume in a subject in
need thereof,
said method comprising administering to said subject a therapeutically
effective amount of an
adenosine-A2A (A2A) receptor antagonist and a programmed cell death protein 1
(PD-1)
signaling pathway inhibitor.
[0534] Embodiment 77. A method of decreasing tumor volume in a subject in
need thereof,
said method comprising administering to said subject a therapeutically
effective amount of an
adenosine-A2A (A2A) receptor antagonist, wherein said A2A receptor antagonist
is a compound
of folinula:
RI
N
Na
N
R3 (1),
wherein,
R1 is independently hydrogen, halogen, -CV3, -CN, -S02C1, -SO0R9,
_m_p4H2, _oNR9R10, _NHC=(0)NHNH2, -NHC=(0)NR9Rio,
-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9R1o, -0R9, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl;
R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -S0n2R11,
-S0v2NR _NHNH2, _0NRI _NHc=(3)NHNH2,
-NHC =(0)NR IR12, _N(0)m2, NR" R'2, _NH_o_Ri 1, _C (0)-0Ri 1,
_c(0)NRIIR12,
-0R11, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R3 is independently hydrogen, halogen, -CX'3, -CN, -S02C1, -S0,13R13,
-S0,3NR _NHNH2, _0NRI3R14, _
NHC=(0)NHNH2, -NHC=(0)NR13R14,
_N(0)m3,

_NR13R14, _c(o)R13, _C(0)-0R13, -C(0)NR13R14, _orsIC 13, = 1
substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
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cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl;
R9, RH), RH., R12, R13 and R'4
are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -802C1, -803H, -804H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NIIC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl;
Xb and X' are independently ¨F, -Cl, -Br, or ¨I;
ni, n2 and n3 are independently an integer from 0 to 4;
ml, m2 and m3 are independently an integer from 1 to 2; and
Vi, v2 and v3 are independently an integer from 1 to 2.
[0535] Embodiment 78. The method of embodiment 77, wherein said A2A
receptor
antagonist is a compound of formula:
N,
Ni\TH2
0
[0536] Embodiment 79. The method of embodiment 77 or 78, further comprising

administering a therapeutically effective amount of a PD-1 signaling pathway
inhibitor.
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[0537] Embodiment 80. The method of embodiment 79, wherein said PD-1
signaling
pathway inhibitor is a PD-Li antagonist.
[0538] Embodiment 81. The method of embodiment 80, wherein said PD-Li
antagonist is a
small molecule or an antibody.
[0539] Embodiment 82. A method of enhancing anti-tumor immune memory in a
subject in
need thereof, said method comprising administering to said subject a
therapeutically effective
amount of an adenosine-A2A (A2A) receptor antagonist and a programmed cell
death protein 1
(PD-1) signaling pathway inhibitor.
[0540] Embodiment 83. A method of enhancing anti-tumor immune memory in a
subject in
need thereof, said method comprising administering to said subject a
therapeutically effective
amount of an adenosine-A2A (A2A) receptor antagonist, wherein said A2A
receptor antagonist
is a compound of formula:
RI
N
Na
2
R3 (I),
wherein,
R1 is independently hydrogen, halogen, -CXa3, -CN, -S02C1, -SOr1R9,
_NHNH2, _0NR9Rio, _NHc=(0)NHNH2, _NHc=(0)NR9Rio, _N(0).1,_NR9Rio,
-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0)NR9R1o, -0R9, substituted or unsubstituted
alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl;
R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -S001R11,
-S0,2NR11R12, ¨NHNH2, ¨0NR11R12, ¨NHC=(0)NHNH2,
¨NHC=(0)NR IR12, _N(0).2, NR' 1R12, _C(0)-0R11, -C(0)NR11R12,
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-OR", substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R3 is independently hydrogen, halogen, -CX'3, -CN, -S02C1, -S0n3R13,
-S0,3NRI3R14, _NHNH2, _0NRI3R14, _NHc=(0)NHNH2, _NHc=(0)NRI3R14,
-N(0).3, -NRi3Ri4, _NH_O_Ri3, _
C(0)R13, -C(0)-0R13, -C(0)NR13R14, _oR13, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl;
R9, Ric), Ri2, Ri3 and R'4
are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NHNH2, -0NH2, -NTIC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl;
)0, Xb and X' are independently ¨F, -Cl, -Br, or ¨I;
ni, n2 and n3 are independently an integer from 0 to 4;
mi., m2 and m3 are independently an integer from Ito 2; and
Vi, v2 and v3 are independently an integer from 1 to 2.
[0541] Embodiment 84. The method of embodiment 83, wherein said A2A
receptor
antagonist is a compound of formula:
181

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\ N N H2
____________________ N
0
[0542] Embodiment 85. The method of embodiment 83 or 84, further comprising

administering a therapeutically effective amount of a PD-1 signaling pathway
inhibitor.
[0543] Embodiment 86. The method of embodiment 85, wherein said PD-1
signaling
pathway inhibitor is a PD-Li antagonist.
[0544] Embodiment 87. The method of embodiment 86, wherein said PD-Li
antagonist is a
small molecule or an antibody.
[0545] Embodiment 88. A method of increasing global immune activation in a
subject in
need thereof, said method comprising administering to said subject a
therapeutically effective
amount of an adenosine-A2A (A2A) receptor antagonist, wherein said A2A
receptor antagonist
is a compound of formula:
N/
\lµ R
R3 (D,
wherein,
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RI is independently hydrogen, halogen, -CV3, -CN, -S02C1, -SOralt9,
-S0,1NR9Rto, _NHNH2, _0NR9R10, _NHc=(0)NHNH2, _NHc=(0)NR9Rto, _
N(0)õ,t,-
NR9Rto,
-NH-O-R9, -C(0)R9, -C(0)-0R9, -C(0 )NR9- Kto, -OR 9, substituted or
unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl;
R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -S0,12R11,
-S0,2NRitR12, NHNH2, 0NR ttR12, NHic (0)NHNH2,
-NHC-(0)NR C(0)-OR", -C(0)NRI1R12,
-OR", substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R3 is independently hydrogen, halogen, -CXc3, -CN, -S02C1, -S0,13R13,
-S0,3NR13R14, _NHNH2, _0NRI3R14, _NHc=(0)NHNH2, _NHc=(0)NR13R14,
-N(0)n-,3, -NR13R14,NH-0-1e, -C(0)R13, -C(0)-0R13, -C(0)NRt3R14, , =
,
suostautea or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl;
R9, Rto, RH., R12, R13 and R'4
are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl;
Xb and X' are independently -F, -Cl, -Br, or -I;
nt, n2 and n3 are independently an integer from 0 to 4;
mt, m2 and m3 are independently an integer from 1 to 2; and
vi, v2 and v3 are independently an integer from 1 to 2.
[0546] Embodiment 89. The
method of embodiment 88, wherein said A2A receptor
antagonist is a compound of formula:
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N
\ NN N H2
____________________ N
0
[0547] Embodiment 90. The method of embodiment 88 or 89, further comprising

administering a therapeutically effective amount of a PD-1 signaling pathway
inhibitor.
[0548] Embodiment 91. The method of embodiment 90, wherein said PD-1
signaling
pathway inhibitor is a PD-Li antagonist.
[0549] Embodiment 92. The method of embodiment 91, wherein said PD-Li
antagonist is a
small molecule or an antibody.
[0550] Embodiment 93. The method of one of embodiments 88-92, wherein said
method
comprises activating a CD4 T cell in said subject.
[0551] Embodiment 94. The method of embodiment 93, wherein said CD4 T cell
is a
memory T cell.
[0552] Embodiment 95. The method of embodiment 93, wherein said CD4 T cell
is an
effector T cell.
[0553] Embodiment 96. The method of one of embodiments 88-95, wherein the
relative
amount of CD45RA-negative CD4 T cells in said subject is increased.
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[0554] Embodiment 97. The method of one of embodiments 88-95, wherein the
relative
amount of CD4 T cells in said subject is increased.
[0555] Embodiment 98. The method of one of embodiments 88-95, wherein the
relative
amount of memory T cells in said subject is increased.
[0556] Embodiment 99. The method of one of embodiments 88-95, wherein the
relative
amount of effector T cells in said subject is increased.
[0557] Embodiment 100. The method of one of embodiments 88-95, wherein said
method
comprises increasing the number of PD-1 positive cells in said subject.
[0558] Embodiment 101. The method of one of embodiments 88-92, wherein said
method
comprises activating a CD8 T cell in said subject.
[0559] Embodiment 102. The method of embodiment 101, wherein the relative
amount of
CD8 T cells in said subject is increased.
[0560] Embodiment 103. The method of one of embodiments 88-102, wherein the
relative
frequency of TCR recombination is increased.
[0561] Embodiment 104. The methods of one of embodiments 1, 23, 64, 65, 70,
71, 76, 77,
82, 83 or 88 wherein said subject is an anti-PD-1 refractory subject.
[0562] Embodiment 105. The method of one of embodiments 1, 26, 64, 67, 76 or
79,
wherein said A2A receptor antagonist is administered at an amount of about 100
mg BID.
[0563] Embodiment 106. The method of embodiment 105, wherein said A2A receptor

antagonist is administered for 28 consecutive days.
[0564] Embodiment 107. The method of one of embodiments 1, 26, 64, 67, 76, 79,
105 or
106, wherein said PD-1 signaling pathway inhibitor is administered at an
amount of about 840
mg.
[0565] Embodiment 108. The method of any one of embodiments 105-107, wherein
said
A2A receptor antagonist is administered at a first time point and said PD-1
signaling pathway
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inhibitor is administered at a second time point, wherein said first time
point precedes said
second time point.
[0566] Embodiment 109. The method of embodiment 108, wherein said second time
point is
within less than about 120, 90, 60, 50, 40, 30, 28, 20, 19, 18, 17, 16, 15,
14, 13, 12, 10, 11,9, 8,
7, 6, 5, 4, 3, 2 or 1 days from said first time point.
[0567] Embodiment 110. The method of embodiment 108 or 109, wherein said
second time
point is within about 14 or 28 days from said first time point.
[0568] Embodiment 111. The method of one of embodiments 1, 23, 64 or 65, said
method
comprising activating a T cell in said subject.
[0569] Embodiment 112. The method of one of embodiments 1, 23, 64 or 65, said
method
comprising inhibiting A2A receptor activity of a cell in said subject.
[0570] Embodiment 113. The method of one of embodiments 1, 23, 64 or 65, said
method
comprising increasing an anti-tumor immune response in a subject.
[0571] Embodiment 114. The method of one of embodiments 1, 23, 64 or 65, said
method
comprising increasing the amount of CD8-positive cells relative to the amount
of regulatory T
cells in said subject.
[0572] Embodiment 115. The method of one of embodiments 1, 23, 64 or 65, said
method
comprising enhancing anti-tumor immune memory in said subject.
[0573] Embodiment 116. The method of one of embodiments 1, 23, 64 or 65, said
method
comprising increasing global immune activation in said subject.
[0574] Embodiment 117. A pharmaceutical composition comprising an A2A receptor

antagonist, a PD-1 signaling pathway inhibitor and a pharmaceutically
acceptable excipient.
[0575] Embodiment 118. The pharmaceutical composition of embodiment 117,
wherein said
A2A receptor antagonist is a compound of formula:
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RI
2
R3 (0,
wherein,
is independently hydrogen, halogen, -CXa3, -CN, -S02C1, -SOõIR9,
-S0,4NR9Rio, _NHNH2, _0NR9Rio, _NHc=.(0)NHNH2, _NHc=,(0)NR9Rio, _
N(0)õ,1,-
NR9Rio,
-C(0)R9, -C(0)-0R9, -C(0 )NR9- Kio, -OR 9, substituted or unsubstituted alkyl,

substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl;
R2 is independently hydrogen, halogen, -CXb3, -CN, -S02C1, -S0n2R11,
-S0,2NRI1R12, _NHNH2, tR12, _
NHC-(0)NHNH2,
-NHC-(0)NR C(0)-OR", -C(0)NR11R12,
-OR", substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R3 is independently hydrogen, halogen, -CX'3, -CN, -S02C1, -S0õ3R13,
-S0,3NR13R14, _NHNH2, _oNRI3R14, _NHc=(0)NHNH2, _NHc=(0)NRI3R14,
-N(0)1,3, -NR13R14, -NH-O-R13, -C(0)R13, -C(0)-0R13, -C(0)NRI3R14, _0- i3, ,
,
suDstitutea or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl;
R9, Rio, RI.% R12, RD and R'4
are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO4H, -SO2NH2,
-NO2, -NI-I2, -NHNH2, -ONH2, -NTIC=(0)NtINH2, substituted or unsubstituted
alkyl, substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl;
Xa, Xb and X' are independently -F, -Cl, -Br, or -I;
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n1, n2 and n3 are independently an integer from 0 to 4;
ml, m2 and m3 are independently an integer from 1 to 2; and
Vi, v2 and v3 are independently an integer from 1 to 2.
[0576] Embodiment 119. The phaimaceutical composition of embodiment 118,
wherein said
A2A receptor antagonist is a compound of formula:
RI
N
Na
N NNH
R6 2
-Re'
R'1 (II),
wherein
R6, R6I and R6.2 are independently hydrogen, halogen, =0, =S, -CF3,
-CN, -CC13, -COOH, -CH2COOH, -CONH2, -OH, -SH, -S02C1, -S03H, -SO2NH2,
-NO2, -NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, substituted or unsubstituted alkyl,
substituted
or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted heteroaryl.
[0577] Embodiment 120. The pharmaceutical composition of one of embodiments
117-119,
wherein said A2A receptor antagonist is a compound of formula:
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\ N N H2
-N
0
[0578] Embodiment 121. The phaimaceutical composition of one of embodiments
117-120,
wherein said PD-1 signaling pathway inhibitor is a programmed death-ligand 1
(PD-L1)
antagonist or a PD-1 antagonist.
[0579] Embodiment 122. The pharmaceutical composition of embodiment 121,
wherein said
programmed death-ligand 1 (PD-L1) antagonist is an antibody or a small
molecule.
[0580] Embodiment 123. The pharmaceutical composition of embodiment 121,
wherein said
PD-Li antagonist is an antibody.
[0581] Embodiment 124. The pharmaceutical composition of embodiment 122 or
123,
wherein said antibody is atezolizumab.
[0582] Embodiment 125. The phaintaceutical composition of one of embodiments
121-124,
wherein said PD-1 antagonist is an antibody or a small molecule.
[0583] Embodiment 126. The phaintaceutical composition of one of embodiments
117-125,
wherein said A2A receptor antagonist and said PD-1 signaling pathway inhibitor
are present in a
combined synergistic amount, wherein said combined synergistic amount is
effective to treat
cancer in a subject in need thereof.
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EMBODIMENTS II
[0584] Embodiment 1. A method of detecting a phosphorylated cAMP response
element-
binding protein (pCREB) in a B-cell or T-cell of a mammalian subject, said
method comprising:
(i) obtaining a blood sample from a mammalian subject;
(ii) contacting said blood sample with an adenosine receptor agonist;
(iii) contacting said blood sample with a pCREB detection agent and a blood
cell
detection agent, wherein said blood cell detection agent comprises a B-cell
detection agent or T-cell detection agent, thereby follning a T-cell-detection
agent
complex or a B-cell-detection agent complex; and
(iv) detecting said T-cell detection agent complex or said B-cell detection
complex
thereby detecting said pCREB in a T-cell or B-cell.
[0585] Embodiment 2. The method of embodiment 1, wherein the adenosine
receptor
agonist comprises adenosine, 5'-N-Ethylcarboxamidoadenosine (NECA), or an
analog thereof.
[0586] Embodiment 3. The method of any of embodiments 1 or 2, wherein the
pCREB
detection agent comprises an antibody against pCREB,
[0587] Embodiment 4. The method of any of embodiments 1-3, wherein the B
cell
detection agent comprises an antibody against CD19 and/or an antibody against
CD2O.
[0588] Embodiment 5. The method of any of embodiments 1-4, wherein the T
cell
detection agent comprises an antibody against CD3, CD4 and/or an antibody
against CD8.
[0589] Embodiment 6. The method of any of embodiments 1-5, further
comprising
contacting said blood sample with a fixation agent and cell permeabilizing
agent after contacting
the blood sample with an adenonsine receptor agonist and prior to contacting
said blood sample
with a pCREB detection agent.
[0590] Embodiment 7. The method of any of embodiments 1-6, further
comprising
contacting said blood sample with an apoptotic cell detection agent.
[0591] Embodiment 8. The method of embodiment 7, wherein the apoptotic cell
detection
agent comprises an antibody against cPARP.
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[0592] Embodiment 9. The method of any of embodiments 1-8, further
comprising, prior
to obtaining said blood sample, administering to said mammalian subject an
adenosine receptor
antagonist
[0593] Embodiment 10. The method of embodiment 9, wherein said adenosine
receptor
antagonist comprises an A2a receptor antagonist or an A2b receptor antagonist.
[0594] Embodiment 11. The method of embodiments 1-10, further comprising,
prior to
obtaining said blood sample, administering to said mammalian subject an anti-
cancer agent.
[0595] Embodiment 12. The method of embodiment 11, wherein said anti-cancer
agent
comprises a PD-Li antagonist.
[0596] Embodiment 13. The method of embodiment 12, wherein said PD-Li
antagonist
comprises atezolizumab.
[0597] Embodiment 14. The method of any of embodiments 1-13, further
comprising
contacting said blood sample with a cell subset detection agent.
[0598] Embodiment 15. The method of embodiment 14, wherein the cell subset
detection
agent comprises a naive cell detection agent, a memory cell detection agent,
or an effector cell
detection agent.
[0599] Embodiment 16. The method of embodiment 14, wherein said cell subset
detection
agent comprises an antibody against CD27 or an antibody against CD45RA.
[0600] Embodiment 17. The method of any one of embodiments 1-16, wherein
said blood
sample is collected from circulating blood.
[0601] Embodiment 18. The method of any one of embodiments 1-16, wherein
said blood
sample comprises an intratumoral sample.
[0602] Embodiment 19. A method of treating a subject with cancer, said
method
comprising:
(i) obtaining a blood sample from a subject with cancer;
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(ii) detecting a level of pCREB induced by an adenosine receptor
agonist in said
sample;
(iii) administering an effective amount of an adenosine receptor
antagonist to said
subject.
[0603] Embodiment 20. The method of embodiment 19, wherein said detecting
of said
level of pCREB induced in said sample comprises:
(a) contacting said blood sample with an adenosine receptor agonist; and
(b) contacting said blood sample with a pCREB detection agent and a blood cell
detection
agent, wherein said blood cell detection agent comprises a B-cell detection
agent or T-
cell detection agent.
[0604] Embodiment 21. The method of embodiment 20, wherein the pCREB
detection
agent comprises an antibody against pCREB.
[0605] Embodiment 22. The method of embodiment 20, wherein the B cell
detection agent
comprises an antibody against CD19 and/or against CD20.
[0606] Embodiment 23. The method of embodiment 20, wherein the T cell
detection agent
comprises an antibody against CD3, CD4 and/or an antibody against CD8.
[0607] Embodiment 24. The method of embodiments 20-23, wherein said
detecting said
level of pCREB induced in said subject comprises measuring a level of pCREB in
B cells or T
cells prior to said administering of the effective amount of an adenosine
receptor antagonist to
said subject.
[0608] Embodiment 25. The method of embodiment 24, further comprising:
(iv) detecting a level of pCREB induced in said sample following said
administering of
the effective amount of adenosine receptor antagonist to said subject.
[0609] Embodiment 26. The method of embodiment 25, wherein said detecting
of the level
of pCREB induce in said sample comprises measuring a level of pCREB induced in
B cells or T
cells following said administering of the effective amount of adenosine
receptor antagonist to
said subject.
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[0610] Embodiment 27. The method of embodiment 26, further comprising
increasing a
dose of an adenosine receptor antagonist based on the level of pCREB induced
in said B cells.
[0611] Embodiment 28. A permeabilized blood cell comprising a pCREB detection
agent and
a blood cell detection agent, wherein said blood cell detection agent
comprises a B-cell detection
agent or T-cell detection agent and said permeabilized blood cell comprises a
permeabilized B-
cell or permeabilized T-cell.
[0612] Embodiment 29. The permeabilized blood cell of embodiment 28,
further
comprising an apoptotic cell detection agent.
[0613] Embodiment 30. The permeabilized blood cell of embodiment 29,
wherein said
apoptotic cell detection agent comprises an antibody against cPARP.
[0614] Embodiment 31. The permeabilized blood cell of embodiment 28,
further
comprising a mature cell detection agent.
[0615] Embodiment 32. The permeabilized blood cell embodiment 31, wherein
said mature
cell detection agent comprises antibody against CD27 or an antibody against
CD45RA.
[0616] Embodiment 33. A container comprising an adenosine receptor agonist
in
combination with the permeabilized cell of embodiment 28.
[0617] Embodiment 34. A flow cytometer comprising the permeabilized blood
cell of
embodiment 28.
EMBODIMENTS III
[0618] Embodiment 1. A method of treating cancer in a subject in need
thereof, said
method comprising administering to said subject a therapeutically effective
amount of an
adenosine-A2A (A2A) receptor antagonist of formula:
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Tsi
NR2
0
0 and a
therapeutically effective amount of
atezolizumab.
1 [0619] Embodiment 2. The method of embodiment 1, wherein said A2A
receptor
2 antagonist and said atezolizumab are administered in a combined
synergistic amount.
1 [0620] Embodiment 3. The method of embodiment 1 or 2, wherein said
adenosine-A2A
2 (A2A) receptor antagonist is administered at 100 mg.
1 [0621] Embodiment 4. The method of one of embodiments 1-3, wherein
said adenosine-
2 A2A (A2A) receptor antagonist is administered twice a day (BID).
1 [0622] Embodiment 5. The method of one of embodiments 1-4, wherein
said
2 atezolizumab is administered at 840 mg.
1 [0623] Embodiment 6. The method of one of embodiments 1-5, wherein
said
2 atezolizumab is administered once every two weeks (Q2W).
1 [0624] Embodiment 7. The method of one of embodiments 1-4 or 6,
wherein said
2 atezolizumab is administered at 1200 mg.
1 [0625] Embodiment 8. The method of one of embodiments 1-7, wherein
said
2 atezolizumab is administered once every three weeks (Q3W).
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1 [0626] Embodiment 9. The method of one of embodiments 1-8, wherein
said cancer is
2 colon cancer, lung cancer, triple negative breast cancer, melanoma, head
and neck cancer,
3 prostate cancer, bladder cancer or renal cancer.
1 [0627] Embodiment 10. The method of one of embodiments 1-9, wherein
said cancer is
2 colon cancer.
1 [0628] Embodiment 11. The method of one of embodiments 1-9, wherein
said cancer is
2 lung cancer.
1 [0629] Embodiment 12. A pharmaceutical composition comprising an
adenosine-A2A
2 (A2A) receptor antagonist of formula:
/N N
\N N 112
_______________________ N
0
3 (,)
and a pharmaceutically acceptable excipient,
4 wherein said adenosine-A2A (A2A) receptor antagonist is present at 100
mg.
1 [0630] Embodiment 13. The pharmaceutical composition of embodiment 12,
further
2 comprising atezolizumab.
1 [0631] Embodiment 14. The pharmaceutical composition of embodiment 13,
wherein said
2 A2A receptor antagonist and said atezolizumab are present at a combined
synergistic amount,
1 [0632] Embodiment 15. The pharmaceutical composition of one of
embodiments 13-14,
2 wherein said atezolizumab is present at 840 mg.
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1 [0633] Embodiment 16. The pharmaceutical composition of one of
embodiments 13-14,
2 wherein said atezolizumab is present at 1200 mg.
1 [0634] Embodiment 17. The pharmaceutical composition of one of
embodiments 12-16,
2 wherein said pharmaceutical composition is an oral dosage form.
1 [0635] Embodiment 18. A pharmaceutical composition comprising an
adenosine-A2A
2 (A2A) receptor antagonist of foimula:
/NIN
NNNH
-N
0
3 0 , atezolizumab and a
pharmaceutically
4 acceptable excipient.
1 [0636] Embodiment 19. The pharmaceutical composition of embodiment 18,
wherein said
2 A2A receptor antagonist and said atezolizumab are present at a combined
synergistic amount.
1 [0637] Embodiment 20. The phaimaceutical composition of embodiment 18
or 19, wherein
2 said A2A receptor antagonist is present at 100 mg.
1 [0638] Embodiment 21. The phaimaceutical composition of any one of
embodiments 18-
2 20, wherein said atezolizumab is present at 840 mg.
1 [0639] Embodiment 22. The phaimaceutical composition of any one of
embodiments 18-
2 20, wherein said atezolizumab is present at 1200 mg.
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1 [0640] Embodiment 23. The pharmaceutical composition of any one of
embodiments 18-
2 22, wherein said pharmaceutical composition is an oral dosage form.
1 [0641] Embodiment 24. A method of treating cancer in a subject in need
thereof, said
2 method comprising administering to said subject a therapeutically
effective amount of an
3 adenosine-A2A (A2A) receptor antagonist of formula:
sN
\NH2
¨N
0
4 0
, wherein said adenosine-A2A (MA) receptor
antagonist is administered at 100 mg twice a day (BID).
1 [0642] Embodiment 25. The method of embodiment 24, wherein said cancer
is colon
2 cancer, lung cancer, triple negative breast cancer, melanoma, head and
neck cancer, prostate
3 cancer, bladder cancer or renal cancer.
1 [0643] Embodiment 26. The method of embodiment 24 or 25, wherein said
cancer is colon
2 cancer.
1 [0644] Embodiment 27. The method of embodiment 24 or 25, wherein said
cancer is lung
2 cancer.
1 [0645] Embodiment 28. The method of one of embodiments 24-27, wherein
said method
2 further comprises administering to said subject a therapeutically
effective amount of
3 atezolizumab.
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1 [0646] Embodiment 29. The method of embodiment 28, wherein said A2A
receptor
2 antagonist and said atezolizumab are administered in a combined
synergistic amount.
1 [0647] Embodiment 30. The method of embodiment 28 or 29, wherein said
atezolizumab is
2 administered at 840 mg.
1 [0648] Embodiment 31. The method of one of embodiments 28-30, wherein
said
2 atezolizumab is administered once every two weeks (Q2W).
1 [0649] Embodiment 32. The method of embodiment 28 or 31, wherein said
atezolizumab is
2 administered at 1200 mg.
1 [0650] Embodiment 33. The method of one of embodiments 28-32, wherein
said
2 atezolizumab is administered once every three weeks (Q3W).
1 [0651] Embodiment 34. A method of activating a T cell, said method
comprising
2 contacting said T cell with an A2A receptor antagonist, wherein said
adenosine-A2A (A2A)
3 receptor antagonist is a compound of formula:
\
Ni
0
4 0
1 [0652] Embodiment 35. The method of embodiment 34, wherein said T cell
is an effector T
2 cell or a natural killer cell.
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1 [0653] Embodiment 36. The method of embodment 34 or 35, wherein said T
cell is an
2 adenosine-suppressed T cell.
1 [0654] Embodiment 37. The method of embodiment 34 or 35, wherein said
T cell is a CD8
2 T cell.
1 [0655] Embodiment 38. The method of embodiment 37, wherein said CD8 T
cell is a
2 CD45RA-negative CD8 Tcell.
1 [0656] Embodiment 39. The method of any one of embodiments 34-38,
wherein said T cell
2 is within a subject.
1 [0657] Embodiment 40. A method of inhibiting A2A receptor activity of
a cell, said method
2 comprising contacting said cell with an A2A receptor antagonist, wherein
said A2A receptor
3 antagonist is a compound of formula:
NI
N H2
_______________________ N
0
4
1 [0658] Embodiment 41. The method of embodiment 40, wherein said
contacting comprises
2 binding said A2A receptor antagonist to an A2A receptor of said cell.
1 [0659] Embodiment 42. The method of any one of embodiments 40-41,
wherein said cell is
2 a T cell.
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1 [0660] Embodiment 43. The method of embodiment 42, wherein said T cell
is an effector T
2 cell or a natural killer cell.
1 [0661] Embodiment 44. The method of embodiment 42, wherein said T cell
is a CD45RA-
2 negative CD8 Tcell.
1 [0662] Embodiment 45. The method of one of embodiments 42-44, wherein
said T cell is
2 within a subject.
1 [0663] Embodiment 46. The method of embodiment 45, wherein said
subject is a cancer
2 subject.
1 [0664] Embodiment 47. The method of embodiment 46, wherein said cancer
subject is an
2 anti-PD-1 refractory subject.
1 [0665] Embodiment 48. A method of increasing an anti-tumor immune
response in a
2 subject in need thereof, said method comprising administering to said
subject a therapeutically
3 effective amount of an adenosine-A2A (A2A) receptor antagonist, wherein
said adenosine-A2A
4 (A2A) receptor antagonist is a compound of formula:
=
7 N H2
-N
0
0
[0666] Embodiment 49. The method of embodiment 48, further comprising
administering a
2 therapeutically effective amount of atezolizumab.
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1 [0667] Embodiment 50. The method of embodiment 48 or 49, wherein said
adenosine-A2A
2 (A2A) receptor antagonist is administered at 100 mg twice a day (BID).
1 [0668] Embodiment 51. The method of any one of embodiments 48-50,
wherein said
2 atezolizumab is administered at 840 mg once every two weeks (Q2W).
1 [0669] Embodiment 52. The method of any one of embodiments 48-50,
wherein said
2 atezolizumab is administered at 1200 mg once every three weeks (Q3W).
1 [0670] Embodiment 53. A method of increasing the amount of CD8-
positive cells relative
2 to the amount of regulatory T cells in a subject in need thereof, said
method comprising
3 administering to said subject a therapeutically effective amount of an
adenosine-A2A (A2A)
4 receptor antagonist, wherein said adenosine-A2A (A2A) receptor antagonist
is a compound of
formula:
N 47N
\
_______________________ 1,1/
0
6 0
1 [0671] Embodiment 54. The method of embodiment 53, further comprising
administering a
2 therapeutically effective amount of atezolizumab.
1 [0672] Embodiment 55. The method of embodiment 53 or 54, wherein said
adenosine-A2A
2 (A2A) receptor antagonist is administered at 100 mg twice a day (BID).
1 [0673] Embodiment 56. The method of any one of embodiments 53-55,
wherein said
2 atezolizumab is administered at 840 mg once every two weeks (Q2W).
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1 [0674] Embodiment 57. The method of any one of embodiments 53-55,
wherein said
2 atezolizumab is administered at 1200 mg once every three weeks (Q3W).
1 [0675] Embodiment 58. A method of decreasing tumor volume in a subject
in need thereof,
2 said method comprising administering to said subject a therapeutically
effective amount of an
3 adenosine-A2A (A2A) receptor antagonist, wherein said adenosine-A2A (A2A)
receptor
4 antagonist is a compound of formula:
N,Nr
\
1\1"2
_______________________ NI
0
0
1 [0676] Embodiment 59. The method of embodiment 58, further comprising
administering a
2 therapeutically effective amount of atezolizumab.
1 [0677] Embodiment 60. The method of embodiment 58 or 59, wherein said
adenosine-A2A
2 (A2A) receptor antagonist is administered at 100 mg twice a day (BID).
1 [0678] Embodiment 61. The method of any one of embodiments 58-60,
wherein said
2 atezolizumab is administered at 840 mg once every two weeks (Q2W).
1 [0679] Embodiment 62. The method of any one of embodiments 58-60,
wherein said
2 atezolizumab is administered at 1200 mg once every three weeks (Q3W).
1 [0680] Embodiment 63. A method of enhancing anti-tumor immune memory
in a subject in
2 need thereof, said method comprising administering to said subject a
therapeutically effective
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3 amount of an adenosine-A2A (A2A) receptor antagonist, wherein said
adenosine-A2A (A2A)
4 receptor antagonist is a compound of formula:
NN
NH2
_______________________ N
0
0
1 [0681] Embodiment 64. The method of embodiment 63, further comprising
administering a
2 therapeutically effective amount of atezolizumab.
1 [0682] Embodiment 65. The method of embodiment 63 or 64, wherein said
adenosine-A2A
2 (A2A) receptor antagonist is administered at 100 mg twice a day (BID).
1 [0683] Embodiment 66. The method of any one of embodiments 63-65,
wherein said
2 atezolizumab is administered at 840 mg once every two weeks (Q2W).
1 [0684] Embodiment 67. The method of any one of embodiments 63-65,
wherein said
2 atezolizumab is administered at 1200 mg once every three weeks (Q3W).
203